Solid-Phase Synthesis of Red Fluorescent Carbon Dots for the Dual-Mode Detection of Hexavalent Chromium and Cell Imaging

The excellent optical properties and biocompatibility of red fluorescence carbon dots (R-CDs) provide a new approach for the effective analysis of hexavalent chromium Cr(VI) in environmental and biological samples. However, the application of R-CDs is still limited by low yield and unfriendly synthesis route. In this study, we developed a new type of R-CDs based on a simple and green solid-phase preparation strategy. The synthesized R-CDs can emit bright red fluorescence with an emission wavelength of 625 nm and also have an obvious visible light absorption capacity. Furthermore, the absorption and fluorescence signals of the R-CDs aqueous solution are sensitive to Cr(VI), which is reflected in color change and fluorescence quenching. Based on that, a scanometric and fluorescent dual-mode analysis system for the rapid and accurate detection of Cr(VI) was established well within the limit of detection at 80 nM and 9.1 nM, respectively. The proposed methods also possess high specificity and were applied for the detection of Cr(VI) in real water samples. More importantly, the synthesized R-CDs with good biocompatibility were further successfully applied for visualizing intracellular Cr(VI) in Hela cells.


Introduction
Heavy metals are a kind of nondegradable, persistent pollutants, and the related research has been the focus and difficulty of biological and environmental science [1,2]. As a typical dangerous metal, hexavalent chromium (Cr(VI)) is widely used in electroplating, leather tanning, textile, chemical fertilizer, stainless steel, welding, and wood preservative industries [3]. Untreated discharge of industrial wastewater and waste residue containing chromium into the environment can lead to the enrichment of Cr(VI) through the food chain, which not only causes serious environmental pollution, but also seriously threatens human life safety [4]. More importantly, a trace amount of Cr(VI) is sufficient to cause bronchitis, skin ulcers, liver, kidney, and nerve tissue damage, and even cancer, [5,6]. The International Agency for Research on Cancer (IARC) classifies chromate as a class substance (human carcinogen). The World Health Organization (WHO) has determined that the maximum pollution level of Cr(VI) in drinking water is 50 µg/L [7], and the United States Environmental Protection Agency (USEPA) has stipulated that the permissible chromium content in drinking water cannot exceed 100 µg/L [8]. In general, the concentration of Scheme 1. The solid-phase synthesis of R-CDs and its applications in dual-mode Cr(VI) detection and cell imaging.

Materials
Aniline hydrochloride, o-phenylenediamine (OPD) and sodium chromate (Na2CrO4) were purchased from Aladdin Biological Technology Co., Ltd. (Shanghai, China). Glycine was obtained from McLean Biological Technology Co., Ltd. (Shanghai, China). Hydrochloric acid (HCl) was purchased from Yantai Yuandong Fine Chemicals Co., Ltd. (Yantai, China). All other reagents were of analytical reagent grade and used without any further purification. Ultrapure water with a resistivity of 18.25 MΩ·cm (UPR-II-40L, Sichuan, China) was used in the whole experiment.

Characterization
The ultraviolet-visible absorption and photoluminescence (PL) spectra of R-CDs were measured by an UV2800S UV-visible spectrophotometer (Shanghai Hengping Scientific Instrument Co., Ltd., Shanghai, China) and an F97Pro fluorescent spectrophotometer (Shanghai Lengguang Technology Co., Ltd., Shanghai, China), respectively. The size distribution and morphology of the prepared R-CDs were characterized by a Tecnai G2 F30 transmission electron microscopy (TEM) with a 200 kV accelerating voltage (FEI, Oregon, USA) and a BI-200SM dynamic light scattering (DLS) particle size analyzer (Brookhaven, New York, USA). X-ray powder diffraction (XRD) spectrum of R-CDs was recorded using a D8-ADVANCE diffractometer (Bruker, Saarbrucken, Germany). The molecular structure of R-CDs was characterized by a NEXUS 670 Fourier transform infrared spectroscopy (FTIR, Nicolet, Wisconsin, USA) and an EscaLab Xi+ X-ray photoelectron spectroscopy (XPS, Thermo Fisher Scientific, Massachusetts, USA). The MTT test was performed on an RT-6100 enzyme-mark analyzer (Shenzhen, China). The fluorescent imaging photographs of the cells were taken using Axioscope A 1 POL fluorescence microscope (ZEISS, Oberkochen, Germany).

Preparation of R-CDs
The R-CDs were synthesized using OPD and aniline hydrochloride as raw materials through a one-step solid-phase synthesis method. Typically, 0.15 g OPD and 0.3 g aniline hydrochloride were grinded thoroughly in an agate mortar, and the resulting mixture Scheme 1. The solid-phase synthesis of R-CDs and its applications in dual-mode Cr(VI) detection and cell imaging.

Materials
Aniline hydrochloride, o-phenylenediamine (OPD) and sodium chromate (Na 2 CrO 4 ) were purchased from Aladdin Biological Technology Co., Ltd. (Shanghai, China). Glycine was obtained from McLean Biological Technology Co., Ltd. (Shanghai, China). Hydrochloric acid (HCl) was purchased from Yantai Yuandong Fine Chemicals Co., Ltd. (Yantai, China). All other reagents were of analytical reagent grade and used without any further purification. Ultrapure water with a resistivity of 18.25 MΩ·cm (UPR-II-40L, Sichuan, China) was used in the whole experiment.

Characterization
The ultraviolet-visible absorption and photoluminescence (PL) spectra of R-CDs were measured by an UV2800S UV-visible spectrophotometer (Shanghai Hengping Scientific Instrument Co., Ltd., Shanghai, China) and an F97Pro fluorescent spectrophotometer (Shanghai Lengguang Technology Co., Ltd., Shanghai, China), respectively. The size distribution and morphology of the prepared R-CDs were characterized by a Tecnai G2 F30 transmission electron microscopy (TEM) with a 200 kV accelerating voltage (FEI, Hillsboro, OR, USA) and a BI-200SM dynamic light scattering (DLS) particle size analyzer (Brookhaven, NY, USA). X-ray powder diffraction (XRD) spectrum of R-CDs was recorded using a D8-ADVANCE diffractometer (Bruker, Saarbrucken, Germany). The molecular structure of R-CDs was characterized by a NEXUS 670 Fourier transform infrared spectroscopy (FTIR, Nicolet, WI, USA) and an EscaLab Xi+ X-ray photoelectron spectroscopy (XPS, Thermo Fisher Scientific, MA, USA). The MTT test was performed on an RT-6100 enzyme-mark analyzer (Shenzhen, China). The fluorescent imaging photographs of the cells were taken using Axioscope A 1 POL fluorescence microscope (ZEISS, Oberkochen, Germany).

Preparation of R-CDs
The R-CDs were synthesized using OPD and aniline hydrochloride as raw materials through a one-step solid-phase synthesis method. Typically, 0.15 g OPD and 0.3 g aniline hydrochloride were grinded thoroughly in an agate mortar, and the resulting mixture was transferred to a 50 mL Teflon-lined stainless steel autoclave and heated in 200 • C for 2 h.
After cooling to room temperature, the product was dispersed in ethanol and dialyzed for 48 h (molecular weight cut-off 7000). The solution was further dried under vacuum conditions, and the obtained solid powder was R-CDs.

Determination of Cr(VI)
The colorimetric and fluorescence detection of Cr(VI) was performed in a 5 mM glycine-hydrochloric acid buffer solution with pH at 2.0 and 3.0, respectively. Amounts of 30 µL Cr(VI) with different concentrations were added to the R-CDs solutions (400 µg/mL), respectively, and incubated for 10 min. The scanometric analysis was carried out in a 96well plate, and RGB values of all solution photos were analyzed by Image J. For fluorescence analysis, the PL spectra were recorded at 560 nm excitation and the PL intensities were recorded at 625 nm. The selectivity of the proposed methods to Cr(VI) was measured by adding various anions and cations, and the detection conditions were the same as described above. The practicability of the methods for Cr(VI) analysis was verified through real water sample analysis and cell imaging.

Cellular Imaging
Hela cells were inoculated on 6-well plates that contained 100 U·mL −1 of penicillin and streptomycin and 10% fetal bovine serum, then cultured in a 5% CO 2 humidified incubator at 37 • C for 48 h. Then, 400 µg·mL −1 R-CDs in glycine-hydrochloric acid buffer solution (5 mM, pH = 3) was used to incubate Hela cells for 20 min in the same culture conditions, and the cells were imaged on a fluorescent microscope after washing twice with fresh buffer solution. Then, different concentrations of Cr(VI) (0, 5, 15, 30 µM) were added and incubated for 10 min. Before imaging, they were washed twice with fresh buffer solution to remove excessive Cr(VI). The images were captured again using a fluorescence microscope under green laser light excitation.

Characterization of R-CDs
The R-CDs were obtained through the facile solid-phase synthesis method, which can avoid the involvement of toxic solvents effectively and further promote the commercial application of R-CDs. The synthesized R-CDs were characterized in detail. As shown in Figure 1a, the UV-visible absorption spectrum of R-CDs displayed three obvious absorption peaks at 285 nm, 560 nm, and 610 nm. The absorption of 285 nm can be assigned to the π-π* transition of C=C/C=N [30] and the absorption peaks at 560 and 610 nm originated from the n-π* transition of C=N and C=O, demonstrating that there is a large-sized conjugated sp2 domain [31]. From Figure S1, it can be seen that the emission wavelength of the R-CDs in aqueous solution does not change with the excitation wavelength change, and the optimal excitation wavelength is at 560 nm. Under the excitation of 560 nm, two emission peaks caused by the large conjugate sp2 domain can be clearly distinguished at 625 nm and 678 nm (Figure 1b). The quantum yield of the R-CDs in ethanol is calculated to be 22.96% by choosing rhodamine B (QY = 56% in ethanol) as reference. The fluorescence of the prepared R-CDs is tolerant against photobleaching and ionic strength even under the continuous irradiation of excitation light for 180 min and the condition of 1 M NaCl concentration ( Figure S2). The particle size distribution and morphology of R-CDs were characterized by DLS and TEM. As can be seen from the DLS data, the particle size of the R-CDs ranged from 2.7 nm to 6.5 nm with an average particle size of 4.0 nm (Figure 2a inset). Spherical R-CDs with good dispersion can be observed clearly from TEM image (Figure 2a). From Figure 1b, we can see that the prepared R-CDs have a wide diffraction peak at about 25 • , indicating that they have an amorphous crystal structure [32,33]. The above results show that the developed R-CDs accord with the general characteristics of typical CDs.  Table S1 shows the values (%) of peak fitted. In summary, the FT-IR and XPS results of R-CDs are consistent. The existence of polar functional groups in R-CDs enables them to disperse well in aqueous solution, which also can promote their interaction with Cr(VI) [36].   C=C/C-C bond, respectively. Then, in the N1s high-resolution spectrum of CDs ( Figure  2e), three peaks appear at 400.2 eV, 399.8 eV, and 398.9 eV, which belong to the pyrrole N, amine N, and pyridine N. The high-resolution O1s spectrum ( Figure 2f) exhibits two different types of O: C=O (532.8 eV) and C-O (531.7 eV). Table S1 shows the values (%) of peak fitted. In summary, the FT-IR and XPS results of R-CDs are consistent. The existence of polar functional groups in R-CDs enables them to disperse well in aqueous solution, which also can promote their interaction with Cr(VI) [36].   The surface-functional groups of the synthesized CDs were characterized by FT-IR. As represented in Figure 2c, the wide peak at 3430 cm −1 is considered as the N-H vibration absorption band [34]. The characteristic peaks at 1620 cm −1 and 1390 cm −1 should be attributed to the stretching vibrations of conjugated structure C=O/C=N and C=C bonds [35]. The absorption peak of C-N bond appeared at 1320 cm −1 , while a peak at 1127 cm −1 is attributed to a C-O stretching vibration. The surface functional groups and elemental composition of R-CDs were further determined by XPS. The full spectrum is shown in Figure S3, the values of the three peaks are 285.11 eV, 400.4 eV, and 531.97 eV, which are assigned to C1s, N1s, and O1s, respectively, and their contents are 80.52%, 15.59%, and 3.91%, respectively. The high-resolution spectrum of C1s ( Figure 2d Table S1 shows the values (%) of peak fitted. In summary, the FT-IR and XPS results of R-CDs are consistent. The existence of polar functional groups in R-CDs enables them to disperse well in aqueous solution, which also can promote their interaction with Cr(VI) [36].

Dual-Mode Detection of Cr(VI)
The as-prepared R-CDs possess perfect optical performance and high-stability and can insure the reasonable sensing application. Inspiringly, the developed R-CDs can be an effective dual-mode sensor for the determination of Cr(VI) based on absorption and fluorescent change. As shown in Figure 3a, the absorption of R-CDs solution varies greatly with a color change from blue to faint yellow after Cr(VI) is involved. To realize simple quantitative analysis, scanometric mode was established based the RGB value of the solution image. The absorption change was described as the ratio value of the sum of G and R to B, and the ratio value changes sharply as shown in Figure 3c. On the other hand, Cr(VI) can also cause the fluorescence intensity decrease in R-CDs, which also can be observed easily from the Figure 3b inset. Moreover, in Figure S4, we can find the signals plateaued after 9 min with increasing reaction time. The effective absorption and fluorescence signal change caused by Cr(VI) lays the foundation of the dual-mode quantitative analysis of Cr(VI) (Figure 3c).
greatly with a color change from blue to faint yellow after Cr(VI) is involved. To realize simple quantitative analysis, scanometric mode was established based the RGB value of the solution image. The absorption change was described as the ratio value of the sum of G and R to B, and the ratio value changes sharply as shown in Figure 3c. On the other hand, Cr(VI) can also cause the fluorescence intensity decrease in R-CDs, which also can be observed easily from the Figure 3b inset. Moreover, in Figure S4, we can find the signals plateaued after 9 min with increasing reaction time. The effective absorption and fluorescence signal change caused by Cr(VI) lays the foundation of the dual-mode quantitative analysis of Cr(VI) (Figure 3c).
The response mechanism of R-CDs to Cr(VI) also was speculated based on a series of experiments, which should be mainly attributed to the unique oxidability of Cr(VI). Firstly, the absorption and fluorescent signals change of R-CDs induced by Cr(VI) must be in acidic conditions (pH = 1~4), and Cr(VI) has a strong oxidizing property at this time. As shown in Figure S5a-c, the involvement of ascorbic acid can significantly hinder the interaction of R-CDs and Cr(VI), and the color, absorption, and fluorescence of R-CDs solution will remain basically unchanged in this case. However, 8-hydroxyquinoline, a strong chelating agent for Cr(VI), has no effect on the dual-mode sensing system after involvement ( Figure S5d-f) [7]. Moreover, the average fluorescence lifetime of R-CDs in the absence and presence of Cr(VI) was 2.41 ns and 0.6 ns, respectively, which indicates the occurrence of dynamic quenching ( Figure S6). Based on the above results, the dual-mode detection process for Cr(VI) can be mainly explained by the oxidation effect of Cr(VI) to R-CDs.  The response mechanism of R-CDs to Cr(VI) also was speculated based on a series of experiments, which should be mainly attributed to the unique oxidability of Cr(VI). Firstly, the absorption and fluorescent signals change of R-CDs induced by Cr(VI) must be in acidic conditions (pH = 1~4), and Cr(VI) has a strong oxidizing property at this time. As shown in Figure S5a-c, the involvement of ascorbic acid can significantly hinder the interaction of R-CDs and Cr(VI), and the color, absorption, and fluorescence of R-CDs solution will remain basically unchanged in this case. However, 8-hydroxyquinoline, a strong chelating agent for Cr(VI), has no effect on the dual-mode sensing system after involvement ( Figure S5d-f) [7]. Moreover, the average fluorescence lifetime of R-CDs in the absence and presence of Cr(VI) was 2.41 ns and 0.6 ns, respectively, which indicates the occurrence of dynamic quenching ( Figure S6). Based on the above results, the dual-mode detection process for Cr(VI) can be mainly explained by the oxidation effect of Cr(VI) to R-CDs.

Analytical Performance
The analytical performance of the proposed dual-mode analysis system was researched carefully. From Figure 4a, the R-CDs solution color changes from blue to faint yellow gradually when increasing the concentration of Cr(VI) that can be distinguished by naked eyes. In this system, 96-well plates were selected as a substrate to support the color reaction and establish the scanometric analysis mode of Cr(VI). Then, the optical photograph was taken in a well-lit area with no obvious shadows by a Smartphone (Huawei HONOR  20). The RGB values of all images taken by a Smartphone were read through the mobile application of Colormeter, and the values of (R + G)/B were calculated to characterize the absorption signal response of R-CDs to different concentrations of Cr(VI). As shown in Figure 4b, the ratio values from the image and the concentrations of Cr(VI) show a good linear relationship in the range of 0.3-50 µM with a correlation coefficient of 0.9971. The limit of detection (LOD) can reach 0.08 µM, which is much lower than the limit value of Cr(VI) in drinking water (~2 µM) [8]. Furthermore, the fluorescent method exhibits higher sensitivity compared to the colorimetric system. From Figure 4c, we can see that the fluorescence intensities of R-CDs present a significant decrease with the increase in Cr(VI) concentrations. The linear range is at 0.03-3 µM between the quenching rate and the concentration of Cr(VI) (r = 0.9965) with a much lower LOD at 9.1 nM, which can ensure the accurate detection of trace Cr(VI) (Figure 4d). More importantly, as shown in Table S2, the sensitivities of developed methods were comparable and even higher than previous reported CDs-based analysis systems of Cr(VI). Therefore, the scanometric analysis mode can be applied for the quantitative detection of Cr(VI) in environmental water samples, and the fluorescent mode can realize the visual fluorescence imaging of Cr(VI) in vivo.

Analytical Performance
The analytical performance of the proposed dual-mode analysis system was researched carefully. From Figure 4a, the R-CDs solution color changes from blue to faint yellow gradually when increasing the concentration of Cr(VI) that can be distinguished by naked eyes. In this system, 96-well plates were selected as a substrate to support the color reaction and establish the scanometric analysis mode of Cr(VI). Then, the optical photograph was taken in a well-lit area with no obvious shadows by a Smartphone (Huawei HONOR 20). The RGB values of all images taken by a Smartphone were read through the mobile application of Colormeter, and the values of (R + G)/B were calculated to characterize the absorption signal response of R-CDs to different concentrations of Cr(VI). As shown in Figure 4b, the ratio values from the image and the concentrations of Cr(VI) show a good linear relationship in the range of 0.3-50 μM with a correlation coefficient of 0.9971. The limit of detection (LOD) can reach 0.08 μM, which is much lower than the limit value of Cr(VI) in drinking water (~2 μM) [8]. Furthermore, the fluorescent method exhibits higher sensitivity compared to the colorimetric system. From Figure 4c, we can see that the fluorescence intensities of R-CDs present a significant decrease with the increase in Cr(VI) concentrations. The linear range is at 0.03-3 μM between the quenching rate and the concentration of Cr(VI) (r = 0.9965) with a much lower LOD at 9.1 nM, which can ensure the accurate detection of trace Cr(VI) (Figure 4d). More importantly, as shown in Table S2, the sensitivities of developed methods were comparable and even higher than previous reported CDs-based analysis systems of Cr(VI). Therefore, the scanometric analysis mode can be applied for the quantitative detection of Cr(VI) in environmental water samples, and the fluorescent mode can realize the visual fluorescence imaging of Cr(VI) in vivo.

Selectivity
The specificity of R-CDs to Cr(VI) was checked systematically to facilitate the practical application of established analysis methods. As shown in Figure 5, common metal 2+ 2+ 2+

Selectivity
The specificity of R-CDs to Cr(VI) was checked systematically to facilitate the practical application of established analysis methods. As shown in Figure 5, common metal ions cannot give rise to obvious color and fluorescence change, including Cu 2+ , Cd 2+ , Zn 2+ , Mg 2+ , Fe 3+ , Fe 2+ , Ni 2+ , Hg 2+ , K+, Cr 3+ , Co 2+ , Ca 2+ , Ag + , and Al 3+ . Only Cr(VI) can cause the color change from blue to faint yellow of R-CDs solution and red fluorescence quenching, as shown in the Figure 5 inset. Furthermore, some common anions also cannot bring irresistible interference to Cr(VI) detection ( Figure S7). All above results indicate that the proposed scanometric and fluorescent method all possesses high-selectivity to meet the requirement of real samples analysis.
12, x FOR PEER REVIEW 8 of color change from blue to faint yellow of R-CDs solution and red fluorescence quenchin as shown in the Figure 5 inset. Furthermore, some common anions also cannot bring i resistible interference to Cr(VI) detection ( Figure S7). All above results indicate that th proposed scanometric and fluorescent method all possesses high-selectivity to meet th requirement of real samples analysis.

Detection of Cr(VI) in Real Water Samples
The application of Cr(VI) detection in environmental water samples was carried ou by using tap water, spring water, and lake water samples, which were obtained from th Qilu University of Technology (Shandong Academy of Sciences) laboratory (Jinan, Ch na), Pearl Spring, and Daming Lake (Jinan, China), respectively. Rainwater was collecte using a wide-mouth container during a rainstorm. All water samples were spiked wi different concentrations Cr(VI) and filtered by using a 0.45 μm filter membrane. Th concentrations of Cr(VI) in the original water samples were all lower than the LOD, an the spiked experiments were performed to evaluate the practicability of the methods. I Table 1, the scanometric analysis mode was first applied for the simple and rapid dete tion of Cr(VI) with spiked concentrations of 1 μM, 10 μM, and 30 μM. The recoverie were between 96.5% and 107.2% and the RSDs were no more than 3.7%. Furthermore, th fluorescence method with a higher sensitivity was used for the trace determination Cr(VI). At the spiked concentrations of 0.1 μM, 0.5 μM, and 1.5 μM, the Cr(VI) could b accurately detected with recoveries of 92.6-107.2% and RSDs lower than 5% (Table S3 Therefore, the established scanometric and fluorescence methods have high practic value for detecting Cr(VI) in environmental samples.

Detection of Cr(VI) in Real Water Samples
The application of Cr(VI) detection in environmental water samples was carried out by using tap water, spring water, and lake water samples, which were obtained from the Qilu University of Technology (Shandong Academy of Sciences) laboratory (Jinan, China), Pearl Spring, and Daming Lake (Jinan, China), respectively. Rainwater was collected using a wide-mouth container during a rainstorm. All water samples were spiked with different concentrations Cr(VI) and filtered by using a 0.45 µm filter membrane. The concentrations of Cr(VI) in the original water samples were all lower than the LOD, and the spiked experiments were performed to evaluate the practicability of the methods. In Table 1, the scanometric analysis mode was first applied for the simple and rapid detection of Cr(VI) with spiked concentrations of 1 µM, 10 µM, and 30 µM. The recoveries were between 96.5% and 107.2% and the RSDs were no more than 3.7%. Furthermore, the fluorescence method with a higher sensitivity was used for the trace determination of Cr(VI). At the spiked concentrations of 0.1 µM, 0.5 µM, and 1.5 µM, the Cr(VI) could be accurately detected with recoveries of 92.6-107.2% and RSDs lower than 5% (Table S3). Therefore, the established scanometric and fluorescence methods have high practical value for detecting Cr(VI) in environmental samples.

RSD (%)
Tap water 1.00 fluorescence method with a higher sensitivity was used for the trace d Cr(VI). At the spiked concentrations of 0.1 μM, 0.5 μM, and 1.5 μM, the accurately detected with recoveries of 92.6-107.2% and RSDs lower than Therefore, the established scanometric and fluorescence methods have value for detecting Cr(VI) in environmental samples.

Intracellular imaging of Cr(VI)
The excellent red fluorescence property and high optical stability R-CDs provide a new chance for visualizing intracellular Cr(VI). MTT was carried out to evaluate the toxicity of R-CDs. From Figure S8, we c survival rate still is over 90% even with the concentration of R-CDs at 800 proves that the R-CDs have a good biocompatibility. As shown in Figur can easily penetrate the cell membrane and enter the cell to emit bright re After adding different concentrations Cr(VI), the fluorescence signal in creased gradually (Figure 6b-d). The result suggests that the proposed used as a new probe for the imaging of intracellular Cr(VI).

Intracellular imaging of Cr(VI)
The excellent red fluorescence property and high optical stability R-CDs provide a new chance for visualizing intracellular Cr(VI). MTT was carried out to evaluate the toxicity of R-CDs. From Figure S8, we c survival rate still is over 90% even with the concentration of R-CDs at 800 proves that the R-CDs have a good biocompatibility. As shown in Figur can easily penetrate the cell membrane and enter the cell to emit bright re After adding different concentrations Cr(VI), the fluorescence signal in creased gradually (Figure 6b-d). The result suggests that the proposed used as a new probe for the imaging of intracellular Cr(VI).

Intracellular Imaging of Cr(VI)
The excellent red fluorescence property and high optical stability of synthesized R-CDs provide a new chance for visualizing intracellular Cr(VI). MTT standard assay was carried out to evaluate the toxicity of R-CDs. From Figure S8, we can see that the survival rate still is over 90% even with the concentration of R-CDs at 800 µg·mL −1 , which proves that the R-CDs have a good biocompatibility. As shown in Figure 6a, the R-CDs can easily penetrate the cell membrane and enter the cell to emit bright red fluorescence. After adding different concentrations Cr(VI), the fluorescence signal in Hela cells decreased gradually (Figure 6b-d). The result suggests that the proposed R-CDs can be used as a new probe for the imaging of intracellular Cr(VI).
was carried out to evaluate the toxicity of R-CDs. From Figure S8, we can see that the survival rate still is over 90% even with the concentration of R-CDs at 800 μg·mL −1 , which proves that the R-CDs have a good biocompatibility. As shown in Figure 6a, the R-CDs can easily penetrate the cell membrane and enter the cell to emit bright red fluorescence. After adding different concentrations Cr(VI), the fluorescence signal in Hela cells decreased gradually (Figure 6b-d). The result suggests that the proposed R-CDs can be used as a new probe for the imaging of intracellular Cr(VI).

Conclusions
In summary, we presented an effective solid-phase synthesis strategy for preparing R-CDs with high fluorescence and stability by simply pyrolyzing the solid-phase precursor of o-phenylenediamine and aniline hydrochloride. The product yield of this reaction can reach 42.4% while avoiding solvent consumption. The synthesized R-CDs can emit bright red fluorescence with a quantum yield of 22.96%. Furthermore, the R-CDs were successfully applied for the sensitive and selective determination of Cr(VI) through a scanometric and fluorescent dual-mode analysis system. The scanometric mode realized the rapid quantitative detection of Cr(VI) in environmental water samples with satisfactory recoveries of 92.6-107.2%, while the fluorescence system was valid for the visualization imaging analysis of Cr(VI) in vivo. The key significance of this work is the green and facile synthesis of R-CDs and the establishment of scanometric/fluorescent dual-mode sensing to Cr(VI) based on single R-CDs, which is conducive to promoting the commercial application of CDs-based nanomaterials.

Conclusions
In summary, we presented an effective solid-phase synthesis strategy for preparing R-CDs with high fluorescence and stability by simply pyrolyzing the solid-phase precursor of o-phenylenediamine and aniline hydrochloride. The product yield of this reaction can reach 42.4% while avoiding solvent consumption. The synthesized R-CDs can emit bright red fluorescence with a quantum yield of 22.96%. Furthermore, the R-CDs were successfully applied for the sensitive and selective determination of Cr(VI) through a scanometric and fluorescent dual-mode analysis system. The scanometric mode realized the rapid quantitative detection of Cr(VI) in environmental water samples with satisfactory recoveries of 92.6-107.2%, while the fluorescence system was valid for the visualization imaging analysis of Cr(VI) in vivo. The key significance of this work is the green and facile synthesis of R-CDs and the establishment of scanometric/fluorescent dual-mode sensing to Cr(VI) based on single R-CDs, which is conducive to promoting the commercial application of CDs-based nanomaterials.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/bios12060432/s1, Figure S1: The PL spectra of R-CDs with excitation wavelength from 400 to 600 nm; Figure S2: Influence of light illumination (a) and ion strength (b) on the PL intensity of R-CDs; Figure S3: XPS survey spectra of synthesized R-CDs; Figure S4: The R, G, B values (a) and fluorescent signal (b) response time of R-CDs to Cr(VI), Inset: pictures of 96-well plates with different response times of Cr(VI); Figure S5: The influence of ascorbic acid (AA) on the scanometric, absorption and fluorescent sensing of Cr(VI), the influence of 8hydroxyquinoline (8-HQ) on the scanometric, absorption and fluorescent sensing of Cr(VI); Figure S6: Fluorescence lifetime of R-CDs and R-CDs-Cr(VI); Figure S7: The change of (R + G)/B (a) values of R-CDs for various anions and the response of luminescence intensity (I 0 − I)/I 0 (b). The colorimetric and fluorescence mode concentrations of all heavy metal anions are 50 µM; Figure S8: MTT test results of R-CDs; Table S1: Values (%) of each peak fitted; Table S2: Comparison of different probes for detection of Cr(VI); Table S3: The fluorescence method is used to detect Cr(VI) in actual water samples.

Data Availability Statement:
The dataset generated and analyzed in this study is not publicly available but may be obtained from the corresponding author upon reasonable request.