# “Turn-On” Fluorescent Assay of Biothiols Based on Nitrogen-Rich Polymer Carbon Nanostrips and Its Application in Cell Imaging

Authors: Xiangying Meng; Jinjuan Qiao; Ronglan Zhao; Hairong Chu; Ying Wang; Xiangyu Chen; Zhengjun Yi
Journal: Journal of Chemistry
 (2019)

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## Abstract

In this work, a sensitive and selective turn-on fluorimetric method has been developed for the determination of biothiols based on blocking Ag+-induced fluorescence quenching of nitrogen-rich polymer carbon nanostrips (NRPCNSs). Ag+ ion can induce the fluorescence quenching of NRPCNSs due to the formation of nonfluorescent coordination complexes via robust Ag-N interaction. Once addition of biothiols, such as cysteine (Cys) and glutathione (GSH), Ag+ ions prefer to interact with biothiols rather than NRPCNSs, which could be attribute to the formation of Ag-S bond, thus leading to effective fluorescent recovery of NRPCNSs. Under the optimized conditions, excellent linear relationships existed between the recovery degree of the NRPCNSs and the concentrations of Cys and GSH in the range of 0.05 μM to 10 μM and 0.2 μM to 30 μM, respectively. And, the limits of detection (LODs) for Cys and GSH are 16.5 nM and 65.1 nM, respectively. The detection system also shows high selectivity against other non-thiol amino acids. Moreover, the potential in practical applications of this proposed method has been demonstrated by detecting biothiols in human serum and fluorescence imaging of biothiols in living cells.


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## Body

## 1. Introduction

Over the years, biological thiols (biothiols), generally refering to cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), have drawn considerable attention due to the essential roles played by these molecules in biological systems [1–3]. Numerous researches have proved that the imbalance of biothiols in biological systems is connected with specific pathological conditions and many human diseases. Specifically, high concentrations of Cys have been linked to neurotoxicity [4], while Cys deficiencies are usually associated with hair depigmentation, slowing growth, leukocyte loss, psoriasis, hematopoiesis decrease, and other body disorders [5, 6]. Elevated concentrations of Hcy are also involved in many diseases, such as cardiovascular diseases, osteoporosis, dementia, and Alzheimer's disease [7]. Among these biothiols, GSH, as the most predominant and abundant intracellular nonprotein thiol, is critical for numerous cellular functions, such as enzyme catalysis, apoptosis, protein synthesis, and transmembrane transport [8]. However, abnormal GSH levels are closely associated with the suppression of immune system functions, acceleration of aging, diabetes, HIV/AIDS disease, and so on [9, 10]. Since the levels of these thiol compounds in human biological samples (such as plasma, live cells, or urine) have important reference value for clinical diagnosis of various diseases [11, 12], it is of great importance and significant interest to develop methods for sensitive, selective, and effective detection of biothiols in biosamples.Up to date, a number of effective analytical techniques have been developed for biothiols detection, typically including high-performance liquid chromatography [13], gas chromatography with mass spectroscopy [14], capillary zone electrophoresis [15], fluorescent determination method [16–22], and so on. Among the methods used, more and more attention has been paid to the fluorescence-based bioanalytical method in recent years due to its several advantages including the high sensitivity, operational simplicity, real-time monitoring, and potential for bioimaging in living systems [23, 24]. As we know, the key issues in the development of fluorimetric method mainly include design of well-performing fluorescent probes. Therefore, a variety of fluorescent probes have been designed for the selective detection of biothiols, including organic dyes [25, 26], heavymetal-based QDs [27], and DNA-based biosensors [28, 29]. Although these probes showed promising results for biothiols detection, some of them are subject to their inherent limitations, such as high cost, toxicity of organic reagents, serious health and environmental concerns, and also tedious synthesis steps [30]. For example, Wu et al. reasonably developed two new quinoline-derived fluorescent probes for the selective discrimination of Cys from GSH/Hcys in the pH 7.4 solutions, and the application for the detection of biothiols in living cells could also be well achieved [31]. Nevertheless, the two probes displayed tedious synthesis steps and requirement of organic reagents.As a promising substitute for some of the aforementioned fluorescent materials, the carbon-based nanomaterials have caused increased research interest, particularly by doping carbon materials with other elements such as nitrogen, sulfur, and boron [32, 33]. Their unique electrical properties, optical properties, and diverse applications make them very strong candidates for the development of the new generation of high-performance fluorimetric method. In recent years, more and more attention has been paid to the application of fluorescent probes in sensing and bioimaging. For instance, Li reported a novel “turn-on” fluorescence probe for sulfide detection based on dihydroxyhemicyanine-Cu2+ complex and was successfully applied to detect sulfide in living cells [34]. Deng’s group designed and synthesized a novel fluorescent nitrogen and sulfur codoped carbon dots by a one-step pyrolysis strategy, which were successfully used for the detection of Hg2+ and fluorescence imaging of biothiols in living cells [35]. Srivastava’s group reported novel N-doped multifluorescent carbon dots for silver and biothiol dual sensing and cell imaging application [36]. The carbon dots displayed high cytocompatibility and without detrimental effect on cellular morphology. Despite these good examples, based on carbon-based nanomaterials, developing more fluorescent probes for effective detection of biothiols in practical application is still needed.Herein, we aimed at developing an efficient “turn-on” fluorescent method for biothiols detection by using N-doped carbon-based nanomaterials as the fluorescent probes. Previous reports have shown that the high affinity of Ag+ to nitrogen elements of the surface of nitrogen-rich polymer carbon nanostrips (NRPCNSs) could effectively quench the fluorescence of the NRPCNSs [33]. It has been also reported that biothiols possess strong affinity toward Ag+ [37, 38]. Inspired by these facts, a novel and simple fluorescent assay for biothiols with Ag+-quenched and biothiols-restored fluorescence intensity of NRPCNSs has been innovatively developed for the first time. Our results show that the as-prepared NRPCNSs possess an excellent water-solubility, uniform morphology, fluorescence stability, and good photoluminescence properties. Fluorescent intensity of the NRPCNSs is quenched by Ag+, and the quenched Ag+-NRPCNSs system can be restored by the addition of biothiols. And, the fluorescence intensity enhancement of the NRPCNSs could be directly related to the amount of biothiol added to the assay solutions. The principle of the proposed method is shown in Scheme 1. Compared with some other previous reports, the developed method is simple, sensitive, and selective for biothiols detection without the complicated synthesis procedure, which was successfully applied to the analysis of biothiols in human serum and living cells.Scheme 1
Schematic diagram of the mechanism of the detection of biothiols by the fluorescent NRPCNSs.


## 2. Materials and Methods

### 2.1. Reagents and Chemicals

Uric acid (UA) was obtained from Sigma-Aldrich. Silver nitrate (AgNO3) was purchased from Nanjing Chemical Reagent Co. Ltd. (Jiangsu, China). L-cysteine (Cys) was purchased from Shanghai Sinopharm Chemical Reagent (Shanghai, China). Glutathione (GSH) was purchased from Aladdin Chemistry (Shanghai, China). SK-hep1 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Phosphate buffer solutions (PBS, pH 6.0–8.0, 50 mM) were prepared by using the different ratios of NaH2PO4 to Na2HPO4. All reagents and solvents were of analytical grade and were used as received without further treatment. All aqueous solutions were prepared with 18 MΩ·cm−1 resistance water, which was obtained from a Milli-Q water purification system.


### 2.2. Apparatus

UV-Vis spectra were tested on a Shimadzu UV-2550 spectrophotometer (Tokyo, Japan). X-ray photoelectron spectroscopy (XPS) analysis was recorded on a Thermo ESCALAB 250 X-ray photoelectron spectrometer (USA). The fluorescence (FL) measurements were conducted on Jasco FP-6500 fluorescence spectrofluorometer (Jasco, Japan). Transmission electron microscopy (TEM) was carried out on a JEM-2100 transmission electron microscope (JEOL Ltd.). FL imaging was performed by TCS SP8 STED 3X (Leica, Germany). FL photographs were taken on the Apple iPhone 6S.


### 2.3. Preparation of Fluorescent NRPCNSs

The fluorescent NRPCNSs were synthesized by a simple hydrothermal method according to previous report with appropriate modifications [33]. In a typical procedure, 1.1 g of UA, 25 mL of ethanol, and 25 mL of deionized water were mixed together to form a homogeneous suspension solution under sonication. Afterwards, 25 mL of the as-prepared mixture was transferred into a stainless steel Teflon-lined autoclave and maintained at 180°C for 4.5 h. After cooling down naturally to room temperature, the obtained products were extracted with dichloromethane. The resulting water phase solution was selected to store in the refrigerator for about one week to remove all large NRPCNSs. Then, the received solution was centrifuged at 8000 rpm for 15 min, and a bright yellow NRPCNSs aqueous solution was finally obtained. The final resultants were stored at 4°C for further use.


### 2.4. Detection Procedure

For the sensing of biothiols, to a 1.5 mL centrifugal tube was sequentially added 50µL of 50 mM PBS (pH 7.4), 5 µL of 2 mg/mL NRPCNSs solution, 40 μL of 1 mM Ag+ ion solution, different amounts of biological thiols solution, and then diluted with deionized water to a volume of 500 µL. After mixing thoroughly on the vortex mixer, the reaction mixture was then incubated at room temperature for 20 min, and the fluorescent spectra of the resulting solution were recorded at 420 nm with the excitation at 355 nm. Both slit widths of the excitation and emission were 3 nm.For selectivity experiments, a series of non-thiol amino acids, including proline (Pro), L-valine (Val), L-tyrosine (Tyr), L-serine (Ser), L-histidine (His), DL-tryptophan (Try), L-arginine (Arg), L-glutamic acid (Glu), L-threonine (Thr), L-Phenylalanine (Phe), L-lysine (Lys), L-α-alanine (Ala), and glycine (Gly) were added into the probe solution (50µL of 50 mM PBS (pH 7.4), 5 µL of 2 mg/mL NRPCNSs solution, and 40 μL of 1 mM Ag+ ion solution in the final test solutions). The concentration of Cys or GSH was 100 μM, and the other interference non-thiol analytes were 1 mM.


### 2.5. Human Serum Sample Preparation

The human serum samples were collected from healthy volunteers in the local hospital. For determination of total content of thiols in human serum samples, the disulfide bonds were firstly reduced to release thiols from its conjugates by addition of triphenylphosphine (PPh3) as reductant [39]. Briefly, 1 mL of the collected human serum samles were vigorously mixed with 80 μL of 0.2 M HCl and 40.0 μL of 0.4 M PPh3 (in water-acetonitrile 20 : 80 v/v and 2 M HCl). After incubating for 20 min under vigorous stirring, the hydrolyzed human serum samples were thoroughly mixed with 1 mL of acetonitrile to precipitate proteins. After the samples were centrifuged at 4000 rpm for 20 min, the supernatants containing the reduced biothiols were collected for subsequent analysis.


### 2.6. Cell Viability Study

The study of cell viability for checking the cytocompatibility of NRPCNSs was conducted using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Briefly, SK-hep1 cells were seeded in 96-well culture plate at a density of 1 × 104 cells per well in 100 μL of Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum in a 5% CO2 environment. After 24 h of seeding, different concentrations of NRPCNSs (mixed with fresh DMEM) were added and incubated with SK-hep1 cells for 24 h. Cells without treatment with NRPCNSs were considered as a control. MTT solution (20 μL, 5 mg/mL) was added to each well, and the cells were incubated for another 4 h. After discarding the culture medium with MTT, 150 μL of DMSO was added and then shaken for 15 min at room temperature. Finally, the absorbance of MTT at 570 nm was examined by a microplate reader (ELx808, Biotek). The cell viability was estimated as the percentage of the absorbance of NRPCNS-treated cells to the absorbance of nontreated cells. All results were repeated for three times.


### 2.7. Cell Imaging Experiment

SK-hep1 cells were cultured in DMEM containing 10% FBS and 1% penicillin streptomycin under a humidified atmosphere containing 5% CO2 at 37°C. Initially, the cells were precultured onto a glass bottom Petri dish with diameter of 20 mm for 24 h, then mixed with NRPCNSs at the final concentration of 40 μg/mL, and incubated for another 8 h prior to cell imaging. The incubated cells were washed three times with pH 7.4 PBS and kept in PBS for imaging by Leica TCS SP8 STED 3X laser scanning confocal microscope.


## 2.1. Reagents and Chemicals

Uric acid (UA) was obtained from Sigma-Aldrich. Silver nitrate (AgNO3) was purchased from Nanjing Chemical Reagent Co. Ltd. (Jiangsu, China). L-cysteine (Cys) was purchased from Shanghai Sinopharm Chemical Reagent (Shanghai, China). Glutathione (GSH) was purchased from Aladdin Chemistry (Shanghai, China). SK-hep1 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Phosphate buffer solutions (PBS, pH 6.0–8.0, 50 mM) were prepared by using the different ratios of NaH2PO4 to Na2HPO4. All reagents and solvents were of analytical grade and were used as received without further treatment. All aqueous solutions were prepared with 18 MΩ·cm−1 resistance water, which was obtained from a Milli-Q water purification system.


## 2.2. Apparatus

UV-Vis spectra were tested on a Shimadzu UV-2550 spectrophotometer (Tokyo, Japan). X-ray photoelectron spectroscopy (XPS) analysis was recorded on a Thermo ESCALAB 250 X-ray photoelectron spectrometer (USA). The fluorescence (FL) measurements were conducted on Jasco FP-6500 fluorescence spectrofluorometer (Jasco, Japan). Transmission electron microscopy (TEM) was carried out on a JEM-2100 transmission electron microscope (JEOL Ltd.). FL imaging was performed by TCS SP8 STED 3X (Leica, Germany). FL photographs were taken on the Apple iPhone 6S.


## 2.3. Preparation of Fluorescent NRPCNSs

The fluorescent NRPCNSs were synthesized by a simple hydrothermal method according to previous report with appropriate modifications [33]. In a typical procedure, 1.1 g of UA, 25 mL of ethanol, and 25 mL of deionized water were mixed together to form a homogeneous suspension solution under sonication. Afterwards, 25 mL of the as-prepared mixture was transferred into a stainless steel Teflon-lined autoclave and maintained at 180°C for 4.5 h. After cooling down naturally to room temperature, the obtained products were extracted with dichloromethane. The resulting water phase solution was selected to store in the refrigerator for about one week to remove all large NRPCNSs. Then, the received solution was centrifuged at 8000 rpm for 15 min, and a bright yellow NRPCNSs aqueous solution was finally obtained. The final resultants were stored at 4°C for further use.


## 2.4. Detection Procedure

For the sensing of biothiols, to a 1.5 mL centrifugal tube was sequentially added 50µL of 50 mM PBS (pH 7.4), 5 µL of 2 mg/mL NRPCNSs solution, 40 μL of 1 mM Ag+ ion solution, different amounts of biological thiols solution, and then diluted with deionized water to a volume of 500 µL. After mixing thoroughly on the vortex mixer, the reaction mixture was then incubated at room temperature for 20 min, and the fluorescent spectra of the resulting solution were recorded at 420 nm with the excitation at 355 nm. Both slit widths of the excitation and emission were 3 nm.For selectivity experiments, a series of non-thiol amino acids, including proline (Pro), L-valine (Val), L-tyrosine (Tyr), L-serine (Ser), L-histidine (His), DL-tryptophan (Try), L-arginine (Arg), L-glutamic acid (Glu), L-threonine (Thr), L-Phenylalanine (Phe), L-lysine (Lys), L-α-alanine (Ala), and glycine (Gly) were added into the probe solution (50µL of 50 mM PBS (pH 7.4), 5 µL of 2 mg/mL NRPCNSs solution, and 40 μL of 1 mM Ag+ ion solution in the final test solutions). The concentration of Cys or GSH was 100 μM, and the other interference non-thiol analytes were 1 mM.


## 2.5. Human Serum Sample Preparation

The human serum samples were collected from healthy volunteers in the local hospital. For determination of total content of thiols in human serum samples, the disulfide bonds were firstly reduced to release thiols from its conjugates by addition of triphenylphosphine (PPh3) as reductant [39]. Briefly, 1 mL of the collected human serum samles were vigorously mixed with 80 μL of 0.2 M HCl and 40.0 μL of 0.4 M PPh3 (in water-acetonitrile 20 : 80 v/v and 2 M HCl). After incubating for 20 min under vigorous stirring, the hydrolyzed human serum samples were thoroughly mixed with 1 mL of acetonitrile to precipitate proteins. After the samples were centrifuged at 4000 rpm for 20 min, the supernatants containing the reduced biothiols were collected for subsequent analysis.


## 2.6. Cell Viability Study

The study of cell viability for checking the cytocompatibility of NRPCNSs was conducted using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Briefly, SK-hep1 cells were seeded in 96-well culture plate at a density of 1 × 104 cells per well in 100 μL of Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum in a 5% CO2 environment. After 24 h of seeding, different concentrations of NRPCNSs (mixed with fresh DMEM) were added and incubated with SK-hep1 cells for 24 h. Cells without treatment with NRPCNSs were considered as a control. MTT solution (20 μL, 5 mg/mL) was added to each well, and the cells were incubated for another 4 h. After discarding the culture medium with MTT, 150 μL of DMSO was added and then shaken for 15 min at room temperature. Finally, the absorbance of MTT at 570 nm was examined by a microplate reader (ELx808, Biotek). The cell viability was estimated as the percentage of the absorbance of NRPCNS-treated cells to the absorbance of nontreated cells. All results were repeated for three times.


## 2.7. Cell Imaging Experiment

SK-hep1 cells were cultured in DMEM containing 10% FBS and 1% penicillin streptomycin under a humidified atmosphere containing 5% CO2 at 37°C. Initially, the cells were precultured onto a glass bottom Petri dish with diameter of 20 mm for 24 h, then mixed with NRPCNSs at the final concentration of 40 μg/mL, and incubated for another 8 h prior to cell imaging. The incubated cells were washed three times with pH 7.4 PBS and kept in PBS for imaging by Leica TCS SP8 STED 3X laser scanning confocal microscope.


## 3. Results and Discussion

### 3.1. The Principle of Biothiol Detection

The NRPCNSs, as an effective fluorescent nanoprobe for determination of biothiols, were prepared by a simple, green, and environmentally friendly hydrothermal method using UA as the carbon-nitrogen source. The synthesis procedure is demonstrated in the experimental section.Scheme1 illustrates the general design of the new turn-on fluorescent response for biothiols. In the absence of biothiols, Ag+ ion, as a kind of strong Lewis acid, has a strong affinity for nitrogen donor atoms [33], which could result in forming the stable metal complexes between Ag+ ion and nitrogen atoms of the surface of NRPCNSs by Ag-N bonds and leading to form nonfluorescent Ag+-NRPCNSs metal complexes by the electron or energy transfer mechanism [40]. Consequently, the fluorescent intensity of the NRPCNSs could be decreased upon the addition of Ag+ ions. On the contrary, in the presence of biothiols, because of a lone pair of electrons from sulfur entered the low-lying vacant d orbitals from silver [41, 42], the Ag-S bond could be formed based on the strong binding preference between Ag+ ion and thiol groups, leaving little or no chance for Ag+ ion binding on the NRPCNSs. Thus, we reasoned that weakly forming Ag+-NRPCNSs metal complexes would display intense fluorescence in the presence of biothiols, and the corresponding turn-on fluorescent response can be used for quantitatively screening of biothiols through fluorescent spectroscopy.To examine the feasibility of our design, the effects of Ag+ ion and the model target, Cys, on the fluorescent intensity of NRPCNSs were investigated. As shown in Figure 1, the NRPCNSs emit highly-intense fluorescence with a maximum at 420 nm (Figure 1(a)). As proposed, when an appropriate amount of Ag+ ion is added to the NRPCNSs solution, the fluorescence is dramatically quenched by Ag+ ions (Figure 1(b)). This clearly demonstrates that the Ag+ ions induces the formation of nonfluorescent Ag+-NRPCNSs coordination complexes by the electron or energy transfer mechanism, thus leading to the quenching of NRPCNSs fluorescence. Interestingly, when Cys is present together with Ag+ ions in the assay solution, the Ag+-induced quenching of NRPCNSs fluorescence does not occur, and the fluorescent intensity recovered to that of free NRPCNSs (Figure 1(c)), indicating that Ag-S bond is much easier to form based on the strong binding preference between Ag+ ion and thiol groups, leaving little or no chance for Ag+ ions binding on the NRPCNSs. Control experiments confirmed that Cys alone cannot induce any changes on the fluorescence of NRPCNSs (Figure 1(d)), which demonstrate that Cys has no significant direct effect on the fluorescence of NRPCNRs. Overall, these observations demonstrated that NRPCNSs together with Ag+ ions can be expected to provide a quantitative readout for biothiols.Figure 1
FL spectra of (a) NRPCNSs (20μg/mL), (b) NRPCNSs (20 μg/mL) + Ag+ ion (80 μM), (c) NRPCNSs (20 μg/mL) + Ag+ ion (80 μM) + Cys (100 μM), and (d) NRPCNSs (20 μg/mL) + Cys (100 μM).


### 3.2. Characterization of the Obtained NRPCNS Nanoprobe

As mentioned above, the NRPCNSs were synthesized from UA by a hydrothermal approach described in the experimental section. Before employing NRPCNSs as the nanoprobe, the UV-Vis spectra, XPS, and morphologies were carefully investigated. As shown in Figure2, the UV-Vis spectrum from the NRPCNSs in aqueous solution (Figure 2(a)) displays three characteristic peaks at ∼275 nm, ∼330 nm, and ∼355 nm, respectively. The absorption peak centered at about 275 nm could be attributed to the π-π∗ transition of aromatic sp2 domains, which results in nearly no observed FL signal [43], while the peak centered approximately at 355 nm leading to strong FL emission at 420 nm (Figure 2(b)) ascribe to the trapping of excited-state energy by the surface states [33]. The peak at about 330 nm with a shoulder peak might be assigned to aromatic polyimides and/or amide intramolecular charge transfer states [33]. Moreover, as shown in the inset of Figure 2, the yellow aqueous solution of NRPCNSs produced intense bright blue fluorescence under the excitation of 365 nm UV light. These results clearly indicate that the as-prepared NRPCNSs can be used as fluorescent nanoprobe for quantitative analysis of biothiols.Figure 2
The UV-Vis spectra of NRPCNRs (a) and the FL spectra of NRPCNRs (b). Inset: photographs of an aqueous solution of the NRPCNRs taken under visible light (1) and 365 nm UV light (2) by means of a mobile phone, respectively.XPS was also employed to characterize the compositions and chemical status of elements in the as-prepared NRPCNSs. As shown in Figure3(a), the full-range XPS spectrum clearly shows three distinct peaks at 284.6, 398.6, and 531.6 eV, corresponding to C1s, N1s, and O1s, respectively. And also, the amounts of C, N, and O elements are 50.53%, 21.11%, and 28.36%, respectively, revealing the presence of C, N, and O as the prominent elements in the as-prepared NRPCNSs. Furthermore, the high-resolution spectrum of C1s is given in Figure 3(b). The C1s peak can be resolved into five components at 288.7, 287.7, 286.2, 285.9, and 284.7 eV, which are attributed to O–C=O, C–N/C=O, C–O, C–N, and C–C/C=C groups, respectively. In the high-resolution spectrum of N1s (Figure 3(c)), three fitted peaks at 401.2, 400.4, and 399.7 eV could be attributed to the graphitic nitrogen, N–H/N–C, and pyridinic nitrogen/pyrrolic nitrogen groups, respectively. The XPS spectrum shows three peaks at 531.3, 531.85, and 532.8 eV in O1s spectrum (Figure 3(d)), attributable to the C=O, C–OH, and C–O–C groups, respectively. As a result, the XPS C1s and N1s spectrum clearly show the formation of pyridinic-like N, pyrrolic-like N, and amino-like N in the NRPCNSs and testify the successful incorporation of N into the NRPCNSs. The abovementioned results suggested that the as-prepared carbon materials are oxygen-doped and nitrogen-rich polymer carbon nanosrips.Figure 3
(a) XPS, (b) C1s, (c) N1s, and (d) O1s spectra of the obtained NRPCNSs.
(a)
(b)
(c)
(d)In order to observe the morphology of the as-prepared fluorescent NRPCNSs and verify the formation of the Ag+-NRPCNSs nanocomposites and the resulting Ag+-NRPCNSs nanocomposites in the presence of Cys, TEM was further performed. As shown in Figure 4, it clearly shows that the TEM images of NRPCNSs are mainly distributed strip structure in the range of 150–180 nm length (Figure 4(a)). As expected, when Ag+ ions are introduced into the NRPCNSs solution, the formation of irregular Ag+-NRPCNSs aggregates can be observed (Figure 4(b)). That is, the surface of the as-prepared NRPCNSs possesses a large amount of nitrogen elements, where they might act as a bridge leading to the NRPCNS aggregation in the presence of Ag+ ions, as a result of the quenching of NRPCNSs FL through the formation of Ag-N bonds. In contrast, no obvious aggregation can be observed upon addition of the mixture of Ag+ and Cys (Figure 4(c)), and this result further confirms that the introduction of Cys disrupts the Ag+-induced NRPCNSs aggregation because of the strong binding preference between the thiol groups and Ag+ ions, which renders the restoration of NRPCNSs FL. These results demonstrate that the designed NRPCNSs-based fluorescent method is suitable for biothiols detection.Figure 4
TEM images of (a) the NRPCNSs, (b) the Ag+-NRPCNSs, and (c) the Ag+-NRPCNSs in the presence of Cys.
(a)
(b)
(c)


### 3.3. Optimization of the Biosensor

To achieve the best analytical performance, the concentration of Ag+ ion and the effect of pH were investigated as follows.The concentration of Ag+ ion plays an important role in the sensing process. That is, the appropriate degree of quenching efficiency by Ag+ ions would lead to a high signal-to-back ground ratio and good sensitivity for biothiols detection. In order to study the quenching behavior of Ag+ ions on the FL of NRPCNSs, the fluorescent signals toward different concentrations of Ag+ ions were investigated first. The fluorescent intensities of NRPCNSs in the absence and presence of Ag+ ions are denoted by FL0 and FL, respectively. As shown in Figure 5(a), as the concentration of Ag+ ions increased, the degree of FL quenching significantly increased with the concentration from 0 µM to 100 µM and then tended to be stable at the higher concentration. Nearly 90% of the FL is quenched by the addition of 100 μM Ag+ ions. This can be ascribed to the much greater extent aggregation of NRPCNSs with the increased amount of Ag+ ions added to the solution. It is worth noting that the use of excessively high concentration of Ag+ ions could result in nonproductive binding of Ag+ ions to the target biothiols. After balancing the quenching efficiency and sensitivity for targets detection, 80 µM was selected as the optimal concentration of Ag+ ions in the biothiol detecting system. More importantly, we observed that Cys could reverse the FL quenching of NRPCNSs by 80 μM Ag+ ions at the greatest extent and return the fluorescent intensity of NRPCNRs to nearly its original value (Figure 1(c)).Figure 5
(a) FL quenching of the NRPCNSs in the presence of different concentrations of Ag+. (b) The FL intensity of the NRPCNSs at different pH values of PBS buffer solution in the absence (purple) or presence of Ag+ (blue) or Ag+ and Cys (red). The concentrations of Ag+ and Cys are 80 µM and 8 μM, respectively.
(a)
(b)The effect of pH in a range between 6.0 and 8.0 was further studied in order to select an appropriate condition for the determination of biothiols with the designed Ag+-NRPCNSs-based fluorescent method. Figure 5(b) shows the fluorescent intensity has a slight change in the pH range between 6.0 and 8.0. Considering the protonation-deprotonation of the NRPCNSs and the high pH can lead to the formation of the insoluble Ag2O from Ag(OH) which formed under strong alkaline conditions, thus leading to incomplete FL quenching and restoration, the mild pH at 7.4 of PBS solution was recommended for use in this work.


### 3.4. Performance of the Fluorescent Assay

Under the optimized conditions (50 mM, pH 7.4 PBS, 20μg/mL NRPCNSs, and 80 μM Ag+ ion), the fluorescent spectra of NRPCNSs in the presence of Cys and GSH with different concentrations were recorded (Figures 6(a) and 6(c)). Figure 6 illustrates that the fluorescent intensity of NRPCNSs at 420 nm gradually increased with increasing concentrations of Cys and GSH. Figure 6(b) displays the fluorescent responses for Cys with varying concentrations from 0 µM to 200 µM, which was proportional to the concentration of Cys. The increased fluorescent values of NRPCNSs exhibited a good linear correlation with the concentration of Cys in the range of 0.05–10 µM (R2 = 0.9931) (inset of Figure 6(b)). The detection limit of Cys was calculated to be about 16.5 nM (S/N = 3), which is lower or comparable to those reported from other fluorescence-based methods [28, 44–48]. The high sensitivity could be attributed to the formation of the robust Ag-S bond. Meanwhile, a good relationship between the increased fluorescence intensity of NRPCNSs and the concentration of GSH in the range of 0.2–30 µM (R2 = 0.9921) is also observed with the detection limit of 65.1 nM (S/N = 3) (Figure 6(d)). It should be noted that the detection of GSH exhibited a relatively low response compared with Cys, which might be attributed to the larger steric hindrance of GSH [49].Figure 6
FL spectra of NRPCNSs in the presence of Ag+ (80 µM) and different concentrations of (a) Cys (0, 0.05, 0.25, 0.5, 1, 3, 5, 8, 10, 20, 40, 60, 100, and 200 µM, bottom to top, excitation at 355 nm) and (c) GSH (0, 0.2, 3, 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, and 200 µM, bottom to top, excitation at 355 nm). Plots of (b) and (d) show the enhanced FL signals ((FL − FL0)/FL0) versus Cys and GSH concentration, respectively. Insets of (b) and (d) show the linear dependence of fluorescence intensities ((FL − FL0)/FL0) on corresponding Cys and GSH concentrations, respectively (error bars represent the standard deviation of three measurements).
(a)
(b)
(c)
(d)


### 3.5. Selectivity of the Detection System

To assess the selectivity of the proposed biosensor for biothiols, various potentially interfering non-thiol amino acids, such as Pro, Val, Tyr, Ser, His, Try, Arg, Glu, Thr, Phe, Lys, Ala, and Gly, were investigated in parallel under the same conditions (50 mM, pH 7.4 PBS, 20μg/mL NRPCNSs, and 80 μM Ag+ ion). As shown in Figure 7, effective prevention of Ag+-induced quenching of NRPCNSs fluorescence takes place only when Cys and GSH are present in the test solutions. In contrast, the other interfering substances only cause the relative fluorescent intensity to change very little even when they are present in concentrations that are 10 times higher than that of Cys and GSH. The above results clearly demonstrate that the proposed detection system shows high selectivity toward biothiols and does not suffer from interference by other non-thiol amino acids, which can be ascribed to the specificity and stable interaction between Ag+ ions and target biothiols. Hence, these observations indicated that the developed program could effective discriminate biothiols from other non-thiol biomolecules.Figure 7
Selectivity of the fluorescent assay for biothiols over other biomolecules. The concentration of Cys or GSH was 100μM, and the other interference non-thiol analytes were 1 mM. The excitation wavelength was 355 nm, and the emission at 420 nm was monitored. Inset shows the corresponding fluorescence spectra of NRPCNSs in the presence of Cys, GSH. and the potential interferences.


### 3.6. Real Sample Analysis

To evaluate practical applications of the developed detection system, the determination of total biothiols content in human serum samples was further performed. Considering that Cys can be bound to proteins or other biothiols through robust S-S bonds in the detection samples [50], the serum samples were pretreated with suitable reducing reagents to release Cys from its conjugates and then used for further analysis. The sample pretreatment procedure is demonstrated in the experimental section. Moreover, it is worth noting that the pretreated serum samples should be firstly diluted by PBS (pH 7.4) to fall into the linear range of the proposed method to obtain quantitative recovery of the spiked biothiols. The total concentration of biothiols in human serum was determined by the standard addition method using Cys as the standard, and the results are shown in Table 1. The recovery results ranged from 96.2% to 104.8% with less than 5.0% relative standard deviation (RSD), indicating that no significant interferences were encountered for the detection of Cys in human serum. The obtained biothiols levels were in good agreement with the reported normal range [47, 51–53]. The analytical characteristics were also compared with other detection methods. As it can be seen in Table 2, the analytical performances are generally better or comparable to those achieved by the previously reported methods. The above results indicate that the developed method possesses great potential for the determination of biothiols in real samples.Table 1
Determination of Cys in human serum samples using the proposed method.
Serum sample
Determined biothiols (μM)
Added Cys (μM)
Measured Cys (μM)
Recovery (%)
RSD (%)
1
331
5
4.86
97.2
4.75
10
9.62
96.2
4.07
2
307
5
5.14
102.8
3.62
10
10.42
104.2
4.95
3
296
5
5.24
104.8
3.19
10
9.94
99.4
2.61Table 2
A comparison of the analytical characteristics of different methods for the detection of biothiols.
Technique
Biothiol
Linear range (μM)
LOD (nM)
Type of sample
Reference
HPLC-ED
Cys
0.1–5
20
Lyophilized serum
[54]
Hcy
0.1–5
30
GSH
0–2.5
90
HPLC-UV
Hcy
1.25–25
100
Plasma
[55]
ECL
Cys
0.5–50
500
Serum
[56]
Colorimetric assay
GSH
0.2–0.9
35.8
Human serum
[48]
Hcy
0.6–3.0
62.4
Cys
0.4–1.2
21.7
Fluorescence sensor
GSH
0.50–40.0
150
Human serum
[47]
Cys
0.50–30.0
110
Hcy
1.0–45.0
200
Fluorescence sensor
GSH
0.1–40
10
Human serum
[57]
Cys
0.5–30
20
Hcy
0.5–50
40
Fluorescence sensor
Cys
0.05–10
16.5
Human serum
Our work
GSH
0.2–30
65.1
SK-hep1 cells
HPLC: high-performance liquid chromatography; ED: electrochemical detection; UV: ultraviolet; ECL: electrochemiluminescence.


### 3.7. Cell Imaging

In order to evaluate the capability of NRPCNSs for fluorescent assay of intracellular biothiols, live-cell imaging studies were carried out. Firstly, the cytotoxicity of NRPCNSs was evaluated, and the standard MTT assays were performed using SK-hep1 cells. As shown in Figure8, upon exposure to different concentrations of NRPCNSs (10, 20, 40, and 80 μg/mL) for 24 h, the cells remained viable, respectively, compared to the control, even at a reasonably high concentration of NRPCNSs (80 μg/mL); the cell viability value still remained more than 89%. These results suggested that NRPCNSs have essentially low cytotoxicity to the cultured cells under experimental conditions.Figure 8
Viability of SK-hep1 incubated with different concentrations of NRPCNSs (0, 10, 20, 40, 60, and 80μg/mL) for 24 h. Inset (from 1 to 6): visual observation of the corresponding solutions of MTT assay; the background is marked 0. Data are mean ± SD (bars) (n = 3).Considering the low cytotoxicity of NRPCNSs, the feasibility of NRPCNSs to detect biothiols in living cells was further examined. As shown in Figure9, the obvious blue emission was observed from SK-hep1 cells when excited at 405 nm, owing to the fluorescent NRPCNSs. And, the favorable overlay of bright field images and fluorescent images further indicated that the as-prepared NRPCNSs had good membrane-permeability. The low cytotoxicity and good cell-membrane permeability of NRPCNSs could render it applicable for fluorescent assay of biothiols in living cells. To examine this possibility, cells were firstly incubated with the NRPCNSs (40 μg/mL) for 8 h at 37°C (Figure 9(a)), incubated with Ag+ (100 μM) for 30 min at 37°C (Figure 9(b)), and then incubated with Cys (100 μM) for 30 min at 37°C (Figure 9(c)), respectively. Figure 9(b) shows the intracellular fluorescence intensity in the cells treated with 100 μM Ag+ was getting weaker (Figure 9(b)). It can be explained that Ag+ diffused into the cells and interacted with the NRPCNSs. Furthermore, when the cells loaded with Ag+-NRPCNSs were further treated with Cys, strong blue fluorescence was observed again (Figure 9(c)). Thus, these results indicated that NRPCNSs is able to display effective fluorescent response to biothiols in living cells.Figure 9
Confocal fluorescence images of SK-hep1 cells. The images from (a) to (c) were cells incubated with probe NRPCNSs (40μg/mL) for 8 h at 37°C, then incubated with Ag+ (100 μM) for 30 min at 37°C, and then incubated with Cys (100 μM) for 30 min at 37°C, respectively. Fluorescence images were taken at 405 nm excitation.
(a)
(b)
(c)


## 3.1. The Principle of Biothiol Detection

The NRPCNSs, as an effective fluorescent nanoprobe for determination of biothiols, were prepared by a simple, green, and environmentally friendly hydrothermal method using UA as the carbon-nitrogen source. The synthesis procedure is demonstrated in the experimental section.Scheme1 illustrates the general design of the new turn-on fluorescent response for biothiols. In the absence of biothiols, Ag+ ion, as a kind of strong Lewis acid, has a strong affinity for nitrogen donor atoms [33], which could result in forming the stable metal complexes between Ag+ ion and nitrogen atoms of the surface of NRPCNSs by Ag-N bonds and leading to form nonfluorescent Ag+-NRPCNSs metal complexes by the electron or energy transfer mechanism [40]. Consequently, the fluorescent intensity of the NRPCNSs could be decreased upon the addition of Ag+ ions. On the contrary, in the presence of biothiols, because of a lone pair of electrons from sulfur entered the low-lying vacant d orbitals from silver [41, 42], the Ag-S bond could be formed based on the strong binding preference between Ag+ ion and thiol groups, leaving little or no chance for Ag+ ion binding on the NRPCNSs. Thus, we reasoned that weakly forming Ag+-NRPCNSs metal complexes would display intense fluorescence in the presence of biothiols, and the corresponding turn-on fluorescent response can be used for quantitatively screening of biothiols through fluorescent spectroscopy.To examine the feasibility of our design, the effects of Ag+ ion and the model target, Cys, on the fluorescent intensity of NRPCNSs were investigated. As shown in Figure 1, the NRPCNSs emit highly-intense fluorescence with a maximum at 420 nm (Figure 1(a)). As proposed, when an appropriate amount of Ag+ ion is added to the NRPCNSs solution, the fluorescence is dramatically quenched by Ag+ ions (Figure 1(b)). This clearly demonstrates that the Ag+ ions induces the formation of nonfluorescent Ag+-NRPCNSs coordination complexes by the electron or energy transfer mechanism, thus leading to the quenching of NRPCNSs fluorescence. Interestingly, when Cys is present together with Ag+ ions in the assay solution, the Ag+-induced quenching of NRPCNSs fluorescence does not occur, and the fluorescent intensity recovered to that of free NRPCNSs (Figure 1(c)), indicating that Ag-S bond is much easier to form based on the strong binding preference between Ag+ ion and thiol groups, leaving little or no chance for Ag+ ions binding on the NRPCNSs. Control experiments confirmed that Cys alone cannot induce any changes on the fluorescence of NRPCNSs (Figure 1(d)), which demonstrate that Cys has no significant direct effect on the fluorescence of NRPCNRs. Overall, these observations demonstrated that NRPCNSs together with Ag+ ions can be expected to provide a quantitative readout for biothiols.Figure 1
FL spectra of (a) NRPCNSs (20μg/mL), (b) NRPCNSs (20 μg/mL) + Ag+ ion (80 μM), (c) NRPCNSs (20 μg/mL) + Ag+ ion (80 μM) + Cys (100 μM), and (d) NRPCNSs (20 μg/mL) + Cys (100 μM).


## 3.2. Characterization of the Obtained NRPCNS Nanoprobe

As mentioned above, the NRPCNSs were synthesized from UA by a hydrothermal approach described in the experimental section. Before employing NRPCNSs as the nanoprobe, the UV-Vis spectra, XPS, and morphologies were carefully investigated. As shown in Figure2, the UV-Vis spectrum from the NRPCNSs in aqueous solution (Figure 2(a)) displays three characteristic peaks at ∼275 nm, ∼330 nm, and ∼355 nm, respectively. The absorption peak centered at about 275 nm could be attributed to the π-π∗ transition of aromatic sp2 domains, which results in nearly no observed FL signal [43], while the peak centered approximately at 355 nm leading to strong FL emission at 420 nm (Figure 2(b)) ascribe to the trapping of excited-state energy by the surface states [33]. The peak at about 330 nm with a shoulder peak might be assigned to aromatic polyimides and/or amide intramolecular charge transfer states [33]. Moreover, as shown in the inset of Figure 2, the yellow aqueous solution of NRPCNSs produced intense bright blue fluorescence under the excitation of 365 nm UV light. These results clearly indicate that the as-prepared NRPCNSs can be used as fluorescent nanoprobe for quantitative analysis of biothiols.Figure 2
The UV-Vis spectra of NRPCNRs (a) and the FL spectra of NRPCNRs (b). Inset: photographs of an aqueous solution of the NRPCNRs taken under visible light (1) and 365 nm UV light (2) by means of a mobile phone, respectively.XPS was also employed to characterize the compositions and chemical status of elements in the as-prepared NRPCNSs. As shown in Figure3(a), the full-range XPS spectrum clearly shows three distinct peaks at 284.6, 398.6, and 531.6 eV, corresponding to C1s, N1s, and O1s, respectively. And also, the amounts of C, N, and O elements are 50.53%, 21.11%, and 28.36%, respectively, revealing the presence of C, N, and O as the prominent elements in the as-prepared NRPCNSs. Furthermore, the high-resolution spectrum of C1s is given in Figure 3(b). The C1s peak can be resolved into five components at 288.7, 287.7, 286.2, 285.9, and 284.7 eV, which are attributed to O–C=O, C–N/C=O, C–O, C–N, and C–C/C=C groups, respectively. In the high-resolution spectrum of N1s (Figure 3(c)), three fitted peaks at 401.2, 400.4, and 399.7 eV could be attributed to the graphitic nitrogen, N–H/N–C, and pyridinic nitrogen/pyrrolic nitrogen groups, respectively. The XPS spectrum shows three peaks at 531.3, 531.85, and 532.8 eV in O1s spectrum (Figure 3(d)), attributable to the C=O, C–OH, and C–O–C groups, respectively. As a result, the XPS C1s and N1s spectrum clearly show the formation of pyridinic-like N, pyrrolic-like N, and amino-like N in the NRPCNSs and testify the successful incorporation of N into the NRPCNSs. The abovementioned results suggested that the as-prepared carbon materials are oxygen-doped and nitrogen-rich polymer carbon nanosrips.Figure 3
(a) XPS, (b) C1s, (c) N1s, and (d) O1s spectra of the obtained NRPCNSs.
(a)
(b)
(c)
(d)In order to observe the morphology of the as-prepared fluorescent NRPCNSs and verify the formation of the Ag+-NRPCNSs nanocomposites and the resulting Ag+-NRPCNSs nanocomposites in the presence of Cys, TEM was further performed. As shown in Figure 4, it clearly shows that the TEM images of NRPCNSs are mainly distributed strip structure in the range of 150–180 nm length (Figure 4(a)). As expected, when Ag+ ions are introduced into the NRPCNSs solution, the formation of irregular Ag+-NRPCNSs aggregates can be observed (Figure 4(b)). That is, the surface of the as-prepared NRPCNSs possesses a large amount of nitrogen elements, where they might act as a bridge leading to the NRPCNS aggregation in the presence of Ag+ ions, as a result of the quenching of NRPCNSs FL through the formation of Ag-N bonds. In contrast, no obvious aggregation can be observed upon addition of the mixture of Ag+ and Cys (Figure 4(c)), and this result further confirms that the introduction of Cys disrupts the Ag+-induced NRPCNSs aggregation because of the strong binding preference between the thiol groups and Ag+ ions, which renders the restoration of NRPCNSs FL. These results demonstrate that the designed NRPCNSs-based fluorescent method is suitable for biothiols detection.Figure 4
TEM images of (a) the NRPCNSs, (b) the Ag+-NRPCNSs, and (c) the Ag+-NRPCNSs in the presence of Cys.
(a)
(b)
(c)


## 3.3. Optimization of the Biosensor

To achieve the best analytical performance, the concentration of Ag+ ion and the effect of pH were investigated as follows.The concentration of Ag+ ion plays an important role in the sensing process. That is, the appropriate degree of quenching efficiency by Ag+ ions would lead to a high signal-to-back ground ratio and good sensitivity for biothiols detection. In order to study the quenching behavior of Ag+ ions on the FL of NRPCNSs, the fluorescent signals toward different concentrations of Ag+ ions were investigated first. The fluorescent intensities of NRPCNSs in the absence and presence of Ag+ ions are denoted by FL0 and FL, respectively. As shown in Figure 5(a), as the concentration of Ag+ ions increased, the degree of FL quenching significantly increased with the concentration from 0 µM to 100 µM and then tended to be stable at the higher concentration. Nearly 90% of the FL is quenched by the addition of 100 μM Ag+ ions. This can be ascribed to the much greater extent aggregation of NRPCNSs with the increased amount of Ag+ ions added to the solution. It is worth noting that the use of excessively high concentration of Ag+ ions could result in nonproductive binding of Ag+ ions to the target biothiols. After balancing the quenching efficiency and sensitivity for targets detection, 80 µM was selected as the optimal concentration of Ag+ ions in the biothiol detecting system. More importantly, we observed that Cys could reverse the FL quenching of NRPCNSs by 80 μM Ag+ ions at the greatest extent and return the fluorescent intensity of NRPCNRs to nearly its original value (Figure 1(c)).Figure 5
(a) FL quenching of the NRPCNSs in the presence of different concentrations of Ag+. (b) The FL intensity of the NRPCNSs at different pH values of PBS buffer solution in the absence (purple) or presence of Ag+ (blue) or Ag+ and Cys (red). The concentrations of Ag+ and Cys are 80 µM and 8 μM, respectively.
(a)
(b)The effect of pH in a range between 6.0 and 8.0 was further studied in order to select an appropriate condition for the determination of biothiols with the designed Ag+-NRPCNSs-based fluorescent method. Figure 5(b) shows the fluorescent intensity has a slight change in the pH range between 6.0 and 8.0. Considering the protonation-deprotonation of the NRPCNSs and the high pH can lead to the formation of the insoluble Ag2O from Ag(OH) which formed under strong alkaline conditions, thus leading to incomplete FL quenching and restoration, the mild pH at 7.4 of PBS solution was recommended for use in this work.


## 3.4. Performance of the Fluorescent Assay

Under the optimized conditions (50 mM, pH 7.4 PBS, 20μg/mL NRPCNSs, and 80 μM Ag+ ion), the fluorescent spectra of NRPCNSs in the presence of Cys and GSH with different concentrations were recorded (Figures 6(a) and 6(c)). Figure 6 illustrates that the fluorescent intensity of NRPCNSs at 420 nm gradually increased with increasing concentrations of Cys and GSH. Figure 6(b) displays the fluorescent responses for Cys with varying concentrations from 0 µM to 200 µM, which was proportional to the concentration of Cys. The increased fluorescent values of NRPCNSs exhibited a good linear correlation with the concentration of Cys in the range of 0.05–10 µM (R2 = 0.9931) (inset of Figure 6(b)). The detection limit of Cys was calculated to be about 16.5 nM (S/N = 3), which is lower or comparable to those reported from other fluorescence-based methods [28, 44–48]. The high sensitivity could be attributed to the formation of the robust Ag-S bond. Meanwhile, a good relationship between the increased fluorescence intensity of NRPCNSs and the concentration of GSH in the range of 0.2–30 µM (R2 = 0.9921) is also observed with the detection limit of 65.1 nM (S/N = 3) (Figure 6(d)). It should be noted that the detection of GSH exhibited a relatively low response compared with Cys, which might be attributed to the larger steric hindrance of GSH [49].Figure 6
FL spectra of NRPCNSs in the presence of Ag+ (80 µM) and different concentrations of (a) Cys (0, 0.05, 0.25, 0.5, 1, 3, 5, 8, 10, 20, 40, 60, 100, and 200 µM, bottom to top, excitation at 355 nm) and (c) GSH (0, 0.2, 3, 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, and 200 µM, bottom to top, excitation at 355 nm). Plots of (b) and (d) show the enhanced FL signals ((FL − FL0)/FL0) versus Cys and GSH concentration, respectively. Insets of (b) and (d) show the linear dependence of fluorescence intensities ((FL − FL0)/FL0) on corresponding Cys and GSH concentrations, respectively (error bars represent the standard deviation of three measurements).
(a)
(b)
(c)
(d)


## 3.5. Selectivity of the Detection System

To assess the selectivity of the proposed biosensor for biothiols, various potentially interfering non-thiol amino acids, such as Pro, Val, Tyr, Ser, His, Try, Arg, Glu, Thr, Phe, Lys, Ala, and Gly, were investigated in parallel under the same conditions (50 mM, pH 7.4 PBS, 20μg/mL NRPCNSs, and 80 μM Ag+ ion). As shown in Figure 7, effective prevention of Ag+-induced quenching of NRPCNSs fluorescence takes place only when Cys and GSH are present in the test solutions. In contrast, the other interfering substances only cause the relative fluorescent intensity to change very little even when they are present in concentrations that are 10 times higher than that of Cys and GSH. The above results clearly demonstrate that the proposed detection system shows high selectivity toward biothiols and does not suffer from interference by other non-thiol amino acids, which can be ascribed to the specificity and stable interaction between Ag+ ions and target biothiols. Hence, these observations indicated that the developed program could effective discriminate biothiols from other non-thiol biomolecules.Figure 7
Selectivity of the fluorescent assay for biothiols over other biomolecules. The concentration of Cys or GSH was 100μM, and the other interference non-thiol analytes were 1 mM. The excitation wavelength was 355 nm, and the emission at 420 nm was monitored. Inset shows the corresponding fluorescence spectra of NRPCNSs in the presence of Cys, GSH. and the potential interferences.


## 3.6. Real Sample Analysis

To evaluate practical applications of the developed detection system, the determination of total biothiols content in human serum samples was further performed. Considering that Cys can be bound to proteins or other biothiols through robust S-S bonds in the detection samples [50], the serum samples were pretreated with suitable reducing reagents to release Cys from its conjugates and then used for further analysis. The sample pretreatment procedure is demonstrated in the experimental section. Moreover, it is worth noting that the pretreated serum samples should be firstly diluted by PBS (pH 7.4) to fall into the linear range of the proposed method to obtain quantitative recovery of the spiked biothiols. The total concentration of biothiols in human serum was determined by the standard addition method using Cys as the standard, and the results are shown in Table 1. The recovery results ranged from 96.2% to 104.8% with less than 5.0% relative standard deviation (RSD), indicating that no significant interferences were encountered for the detection of Cys in human serum. The obtained biothiols levels were in good agreement with the reported normal range [47, 51–53]. The analytical characteristics were also compared with other detection methods. As it can be seen in Table 2, the analytical performances are generally better or comparable to those achieved by the previously reported methods. The above results indicate that the developed method possesses great potential for the determination of biothiols in real samples.Table 1
Determination of Cys in human serum samples using the proposed method.
Serum sample
Determined biothiols (μM)
Added Cys (μM)
Measured Cys (μM)
Recovery (%)
RSD (%)
1
331
5
4.86
97.2
4.75
10
9.62
96.2
4.07
2
307
5
5.14
102.8
3.62
10
10.42
104.2
4.95
3
296
5
5.24
104.8
3.19
10
9.94
99.4
2.61Table 2
A comparison of the analytical characteristics of different methods for the detection of biothiols.
Technique
Biothiol
Linear range (μM)
LOD (nM)
Type of sample
Reference
HPLC-ED
Cys
0.1–5
20
Lyophilized serum
[54]
Hcy
0.1–5
30
GSH
0–2.5
90
HPLC-UV
Hcy
1.25–25
100
Plasma
[55]
ECL
Cys
0.5–50
500
Serum
[56]
Colorimetric assay
GSH
0.2–0.9
35.8
Human serum
[48]
Hcy
0.6–3.0
62.4
Cys
0.4–1.2
21.7
Fluorescence sensor
GSH
0.50–40.0
150
Human serum
[47]
Cys
0.50–30.0
110
Hcy
1.0–45.0
200
Fluorescence sensor
GSH
0.1–40
10
Human serum
[57]
Cys
0.5–30
20
Hcy
0.5–50
40
Fluorescence sensor
Cys
0.05–10
16.5
Human serum
Our work
GSH
0.2–30
65.1
SK-hep1 cells
HPLC: high-performance liquid chromatography; ED: electrochemical detection; UV: ultraviolet; ECL: electrochemiluminescence.


## 3.7. Cell Imaging

In order to evaluate the capability of NRPCNSs for fluorescent assay of intracellular biothiols, live-cell imaging studies were carried out. Firstly, the cytotoxicity of NRPCNSs was evaluated, and the standard MTT assays were performed using SK-hep1 cells. As shown in Figure8, upon exposure to different concentrations of NRPCNSs (10, 20, 40, and 80 μg/mL) for 24 h, the cells remained viable, respectively, compared to the control, even at a reasonably high concentration of NRPCNSs (80 μg/mL); the cell viability value still remained more than 89%. These results suggested that NRPCNSs have essentially low cytotoxicity to the cultured cells under experimental conditions.Figure 8
Viability of SK-hep1 incubated with different concentrations of NRPCNSs (0, 10, 20, 40, 60, and 80μg/mL) for 24 h. Inset (from 1 to 6): visual observation of the corresponding solutions of MTT assay; the background is marked 0. Data are mean ± SD (bars) (n = 3).Considering the low cytotoxicity of NRPCNSs, the feasibility of NRPCNSs to detect biothiols in living cells was further examined. As shown in Figure9, the obvious blue emission was observed from SK-hep1 cells when excited at 405 nm, owing to the fluorescent NRPCNSs. And, the favorable overlay of bright field images and fluorescent images further indicated that the as-prepared NRPCNSs had good membrane-permeability. The low cytotoxicity and good cell-membrane permeability of NRPCNSs could render it applicable for fluorescent assay of biothiols in living cells. To examine this possibility, cells were firstly incubated with the NRPCNSs (40 μg/mL) for 8 h at 37°C (Figure 9(a)), incubated with Ag+ (100 μM) for 30 min at 37°C (Figure 9(b)), and then incubated with Cys (100 μM) for 30 min at 37°C (Figure 9(c)), respectively. Figure 9(b) shows the intracellular fluorescence intensity in the cells treated with 100 μM Ag+ was getting weaker (Figure 9(b)). It can be explained that Ag+ diffused into the cells and interacted with the NRPCNSs. Furthermore, when the cells loaded with Ag+-NRPCNSs were further treated with Cys, strong blue fluorescence was observed again (Figure 9(c)). Thus, these results indicated that NRPCNSs is able to display effective fluorescent response to biothiols in living cells.Figure 9
Confocal fluorescence images of SK-hep1 cells. The images from (a) to (c) were cells incubated with probe NRPCNSs (40μg/mL) for 8 h at 37°C, then incubated with Ag+ (100 μM) for 30 min at 37°C, and then incubated with Cys (100 μM) for 30 min at 37°C, respectively. Fluorescence images were taken at 405 nm excitation.
(a)
(b)
(c)


## 4. Conclusions

In this study, an efficient turn-on fluorescent method was successfully developed for the sensitive and selective detection of biothiols based on NRPCNSs as the fluorescent probe. The NRPCNSs with good stability, water solubility, low cytotoxicity, and good cell-membrane permeability were prepared by a facile and green hydrothermal method. In the presence of biothiols, Ag+ preferred to bind to biothiols instead of fluorescent NRPCNSs due to the stronger affinity of Ag+ to biothiols based on the Ag-S bonds, and the fluorescence of NRPCNSs could be restored from the nonfluorescent Ag+-NRPCNSs metal complexes. By measuring the fluorescent intensity changes, the concentrations of target biothiols can be determined. Under the optimal conditions, the method shows high sensitivity and good selectivity over other non-thiol amino acids, and it has also been successfully applied for the determination of biothiols in human serum. Moreover, the as-prepared NRPCNSs can be used for fluorescent bioimaging of biothiols in living cells, which demonstrates its great potential value in practical applications. Nevertheless, a disadvantage of our proposed method is the need for working in the UV (λex = 355 nm), which might display strong background UV absorption in the analysis of biological samples. Thus, the influence of background absorption on detection can be minimized as much as possible by sample pretreatment (e.g., sample dilution, selective precipitation, and protein elimination) in order to obtain good analytical performances.



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*Source: 2847961-2019-01-15.xml*