A Novel “Off-On” Fluorescent Probe Based on Carbon Nitride Nanoribbons for the Detection of Citrate Anion and Live Cell Imaging

A novel fluorescent “off-on” probe based on carbon nitride (C3N4) nanoribbons was developed for citrate anion (C6H5O73−) detection. The fluorescence of C3N4 nanoribbons can be quenched by Cu2+ and then recovered by the addition of C6H5O73−, because the chelation between C6H5O73− and Cu2+ blocks the electron transfer between Cu2+ and C3N4 nanoribbons. The turn-on fluorescent sensor using this fluorescent “off-on” probe can detect C6H5O73− rapidly and selectively, showing a wide detection linear range (1~400 μM) and a low detection limit (0.78 μM) in aqueous solutions. Importantly, this C3N4 nanoribbon-based “off-on” probe exhibits good biocompatibility and can be used as fluorescent visualizer for exogenous C6H5O73− in HeLa cells.

biothiols, and cyanide [18,23,25,26]. However, to the best of our knowledge, there is no report of fluorescent sensors based on C 3 N 4 nanoribbons for citrate anion detection.
In this work, blue fluorescent C 3 N 4 nanoribbons were prepared by alkali-catalyzed hydrolysis from bulk C 3 N 4 [27]. Furthermore, we demonstrated that the obtained C 3 N 4 nanoribbons can serve as a novel "off-on" fluorescent probe for the detection of citrate anion (C 6 H 5 O 7 3− ) with excellent sensitivity and selectivity based on fluorescence quenching by Cu 2+ through a photoinduced electron transfer [28,29] and fluorescence recovery by the addition of C 6 H 5 O 7 3− . We hypothesized that the interaction of Cu 2+ and C 3 N 4 nanoribbons is inhibited by the strong chelation between Cu 2+ and C 6 H 5 O 7 3− [30][31][32]. This is the first time that C 3 N 4 nanoribbons have been applied for C 6 H 5 O 7 3− detection. Importantly, the turn-on fluorescent sensor using this fluorescent "off-on" probe exhibits low cytotoxicity and can be applied for C 6

Characterization
UV-vis spectroscopy measurements were performed on a Shimadzu UV-3600 spectrophotometer. Fluorescence spectra were recorded on an RF-5301PC spectrofluorophotometer. The Fourier transform infrared (FTIR) spectrum of C 3 N 4 nanoribbons was measured on a Nexus 870 FTIR spectrometer. A JEOL 2010 transmission electron microscope (TEM) was used for the characterization of C 3 N 4 nanoribbons. The X-ray diffraction (XRD) pattern of C 3 N 4 nanoribbons was recorded on an X-ray powder diffractometer with graphite monochromatized Cu Kα radiation (D8 Advance; Bruker, Karlsruhe, Germany). The X-ray photoelectron spectroscopy (XPS) investigation was carried out on PHI 5000 VersaProbe system with Al cathode as the X-ray source. The C 3 N 4 nanoribbons was dropped on silicon slices for XPS characterization. Dynamic light scattering (DLS) and zeta potential measurements were conducted on a zeta potential analyzer (Zeta PALS, Brookhaven Instruments Corp., Brookhaven, NY, USA). Cell fluorescent images were recorded by confocal laser scanning microscopy (Olympus FV1000, Tokyo, Japan).

Preparation of C 3 N 4 Nanoribbons
The bulk C 3 N 4 was prepared by the calcination of melamine at 600 • C (5.0 • C min −1 ) for 4 h under an Ar atmosphere [33]. The C 3 N 4 nanoribbons were prepared via the alkali-catalyzed hydrolysis of bulk C 3 N 4 [27]. In brief, 10 mg of bulk C 3 N 4 was dispersed in 10 mL of sodium hydroxide solution (8.0 M) and sonicated for 2 h at 60 • C. After cooling to room temperature, this solution was centrifuged and washed five times with ultrapure water. The product was collected and dialyzed (the molecular weight cutoff of the dialysis bag was 1000 kDa) for further experiments.

Synthesis of Cu 2+ -C 3 N 4 Nanoribbon Complex
One hundred microliters of CuCl 2 (10 mM) was added to 9.9 mL of C 3 N 4 nanoribbons aqueous solution (1 mg mL −1 ). Then, the mixture was stirred for 10 min at room temperature. After that, the mixture was centrifuged and washed with ultrapure water to remove excessive copper ions. Finally, the obtained precipitate was dispersed in ultrapure water to obtain a Cu 2+ -C 3 N 4 nanoribbon solution. were added to the solution of the Cu 2+ -C 3 N 4 nanoribbon complex, respectively. After thoroughly mixing and standing for 20 s, the fluorescence measurements were carried out to investigate the selectivity of the proposed fluorescent sensor.

Cell Imaging and Cytotoxicity Assay
Human cervical cancer (HeLa) cells used in this study were cultured at 37 • C in a 5% CO 2 incubator in DMEM medium, which contains fetal bovine serum (10%), streptomycin (100 mg mL −1 ), and penicillin (100 U mL −1 ). When the cells had grown to 80% confluency, the cells were digested with trypsin, collected and seeded in a confocal dish, and cultured overnight. Then the cells were pretreated with C 6 H 5 O 7 Na 3 (1 mM). After 12 h incubation, the cells were gently washed with phosphate-buffered saline (PBS) solution (10 mM, pH = 7.4) and treated with Cu 2+ -C 3 N 4 nanoribbon complex for another 4 h. Finally, the resulting cells were washed with PBS solution (10 mM, pH = 7.4) and then fluorescence images were taken on a confocal microscope under UV excitation.
For cytotoxicity assay, 100 µL of cells suspension (10 5 cells mL −1 ) was seeded to each well of 96-well plates. Then the medium was replaced by different concentrations of C 3 N 4 nanoribbons or Cu 2+ -C 3 N 4 nanoribbon complex and culture for 24 h. Following this, the cells were washed with PBS solution. After that, the standard MTT assay was carried out for the determination of cell viabilities relative to the untreated cells.

Characterization of C 3 N 4 Nanoribbons
The C 3 N 4 nanoribbons were prepared by ultrasonic exfoliation of bulk C 3 N 4 in an alkaline solution. The related characterizations of bulk C 3 N 4 are shown in Figure S1. TEM images display the morphology and size distribution of the C 3 N 4 nanoribbons. As shown Figure 1a, C 3 N 4 nanoribbons present an average diameter of approximately 5 nm and a length of up to 200 nm. High-resolution transmission electron microscopy (HRTEM) image clearly shows that single and few-layer C 3 N 4 nanoribbons were obtained (Figure 1b). The XRD pattern of C 3 N 4 nanoribbons ( Figure 1c) showed a broad distinct diffraction peak at 27.2 • , which can be ascribed to the strong π-conjugated layers characteristic (002) of C 3 N 4 [17,34]. The composition and structure of C 3 N 4 nanoribbons were confirmed by XPS and FTIR measurements. The XPS survey spectrum of C 3 N 4 nanoribbons ( Figure S2) displays binding energies of C (283 eV) and N (397 eV). The C 1s XPS spectrum of C 3 N 4 nanoribbons ( Figure S3) can be fitted into three peaks centering at 284.6, 285.4, and 287.8 eV, which can be attributed to C-C, sp 2 C=N, and sp 3 C-N of C 3 N 4 , respectively [35][36][37][38][39]. The XPS spectrum of the N 1s spectrum (Figure 1d) can be fitted into three different peaks at 398.3, 399.5, and 400.5 eV, being assigned to C=N-C, (N-(C) 3 ), and -NH 2 , respectively [36,40]. The FTIR spectrum of C 3 N 4 nanoribbons is presented in Figure S4. The broad peaks at 3330 and 3182 cm −1 are ascribed to the stretching vibrations of NH 2 and N-H groups, respectively [39]. The peaks centered at 1637, 1577, 1420, 1334, and 1284 cm −1 indicated the typical stretching modes of CN heterocycles [41][42][43]. Beside the peaks mentioned above, the peak at 810 cm −1 indicated the vibration of the s-triazine ring [34,44].
The photophysical properties of C 3 N 4 nanoribbons were investigated by UV-vis and PL spectra (Figure 2a). The prepared C 3 N 4 nanoribbons present two strong absorption peaks at 216 and 278 nm. Meanwhile, a fluorescent emission at 415 nm can be seen from fluorescence spectrum at the excitation of 360 nm (Figure 2a). The PL intensity of C 3 N 4 nanoribbons increases dramatically with the increasing pH of solution ranging from 9 to 12, and displays slight changes under acid conditions ( Figure S5).

The Influence of Metal Ions on the Fluorescence of C 3 N 4 Nanoribbons
A previous study indicated that Cu 2+ , Fe 3+ , and Ag + can quench the fluorescence of C 3 N 4 due to the photoinduced electron transfer from C 3 N 4 to metal ions [23,25,45]. In this experiment, the fluorescent responses of C 3 N 4 nanoribbons towards different metal ions were investigated. As shown in Figure S6, C 3 N 4 nanoribbons display a slight change of the PL intensity in the presence of Al 3+ , Ba 2+ , K + , Li + , Mg 2+ , Na + , Pb 2+ , Sn 2+ , and Zn 2+ (100 µM). However, the PL intensity of C 3 N 4 nanoribbons dramatically decreases with the addition of Cu 2+ , Ag + , Fe 3+ , Co 2+ , Mn 2+ , and Ni 2+ , especially for Cu 2+ and Ag + . Cu 2+ was chosen as the quencher in this work [36]. A decrease of PL intensity of C 3 N 4 nanoribbons is observed as the concentration of Cu 2+ increases (Figure 2b,c), and it is almost completely quenched after the addition of 40 µM Cu 2+ . Figure 2d shows that the PL intensity of C 3 N 4 at 415 nm versus the concentrations of Cu 2+ exhibits a linear relationship ranging from 10 to 300 nM (R 2 = 0.9976).

Sensitivity and Selectivity of the Fluorescent "Off-On" Probe Based on C 3 N 4 Nanoribbons for C 6 H 5 O 7 3− Detection
The influence of incubation time on the fluorescence recovery of C 3 N 4 nanoribbons was measured. As Figure S7 displayed, the PL intensity of Cu 2+ -C 3 N 4 nanoribbon complex increases rapidly with the time after the addition of C 6 H 5 O 7 3− , and maintains a plateau after 20 s. Therefore, an incubation time of 20 s was selected for subsequent experiments.
As is well known, sensitivity is a critical parameter to assess the sensing performance [46,47]. The sensitivity of the fluorescent sensor using the C 3 N 4 nanoribbon-based "off-on" probe was evaluated. The PL intensity of the Cu 2+ -C 3 N 4 nanoribbon complex with different concentrations of C 6 H 5 O 7 3− was recorded. As illustrated in Figure 3a,b, the PL intensity of the Cu 2+ -C 3 N 4 nanoribbon complex was obviously recovered as the concentration of C 6 H 5 O 7 3− increased. The PL intensity of the Cu 2+ -C 3 N 4 nanoribbon complex was completely recovered with the addition of C 6 H 5 O 7 3− (2.25 mM). The fluorescent sensor using the C 3 N 4 nanoribbon-based "off-on" probe shows a linear range from 1 to 400 µM and the calculated detection limit is 0.78 µM (S/N = 3) (inset in Figure 3b). Compared with previously reported fluorescent probes, such as coumarin [6], rhodamine [13], diketoprrrolopyrrole [15], TPIOP-boronate [48], and CdTe quantum dots [14], the fluorescent sensor using the C 3 N 4 nanoribbon-based "off-on" probe exhibits better sensing performance for C 6 H 5 O 7 3− detection with a broader linear range and faster response time (Table S1) [6,14,15,49]. were also investigated under different pH levels and metal ions environments. As shown in Figure S9a, the fluorescence intensity of the Cu 2+ -C 3 N 4 nanoribbon-based probe with or without C 6 H 5 O 7 3− did not show obvious change in the pH range from 4 to 8. More importantly, almost all of the cations (10 µM) did not affect the fluorescence response of the Cu 2+ -C 3 N 4 nanoribbon-based probe, except for Ag + ( Figure S9b). Further, cell lysate (1 × 10 6 cell mL −1 ) and biological molecules (10 µM), including glutamic acid, ascorbic acid, glutathione, and DNA, were introduced to examine the probe's stability. As Figure S9c shows, except for glutathione, the Cu 2+ -C 3 N 4 nanoribbon-base probe exhibited no obvious fluorescence change upon the addition of C 6 H 5 O 7 3− and a biological molecule (10 µM) mixture, even in cell lysate.
These results indicate that the Cu 2+ -C 3 N 4 nanoribbon-based probe can be used for C 6 H 5 O 7 3− detection in more complex environments. The mechanism of the fluorescence quenching and recovering process were studied by DLS and zeta-potential for this system ( Figure S10). The average hydrodynamic size of C 3 N 4 nanoribbons increased from 230 to 1523 nm in the presence of Cu 2+ along with fluorescence quenching. A change of the zeta-potential from −20.6 mV to −10.4 mV was observed after the C 3 N 4 nanoribbons interacted with Cu 2+ . This result indicates that a non-fluorescent complex (Cu 2+ -C 3 N 4 nanoribbon) was formed [50]. With the addition of C 6 H 5 O 7 3− , the hydrodynamic size decreased because of the chelation between Cu 2+ and C 6 H 5 O 7 3− , indicating the release of Cu 2+ from C 3 N 4 nanoribbons and resulting in the restoration of the fluorescence of C 3 N 4 nanoribbons [18,19,40]. In order to rule out the effect of nonspecific binding, the PL intensity of the C 3 N 4 nanoribbons was monitored after the addition of different anion solutions. The result shows that, except for OH − , the other anions did not dramatically affect the PL intensity of the C 3 N 4 nanoribbons ( Figure S11).

Intracellular Imaging of C 6 H 5 O 7 3−
For further biological applications, the cytotoxicity of the C 3 N 4 nanoribbons and Cu 2+ -C 3 N 4 nanoribbon complex to HeLa cells was assessed through MTT assays. As shown in Figure S12, the viability of HeLa cells showed no obvious change after incubation with the C 3 N 4 nanoribbons or Cu 2+ -C 3 N 4 nanoribbon complex for 24 h, indicating their good biocompatibility. Since the Cu 2+ -C 3 N 4 nanoribbon complex showed a highly selective and sensitive response towards C 6

Conclusions
In summary, a novel fluorescent "off-on" probe based on C 3 N 4 nanoribbons was developed for C 6 H 5 O 7 3− detection. The fluorescence of C 3 N 4 nanoribbons can be quenched by Cu 2+ and then recovered by the addition of C 6 H 5 O 7 3− . The sensor using this fluorescent "off-on" probe showed a good detection linear range (1~400 µM) with a low detection limit (0.78 µM) as well as high selectivity in aqueous solutions. More importantly, this "off-on" probe based on C 3 N 4 nanoribbons exhibited good biocompatibility and low cytotoxicity in cell environments and can be utilized for intracellular imaging of C 6 H 5 O 7 3− .