Cucurbit[6]uril-based carbon dots for recognizing L -tryptophan and capecitabine †

Fluorescent nitrogen and fluorine doped carbon dots (CDs) were prepared by a hydrothermal method using levofloxacin (LVFX) and cucurbit[6]uril (Q[6]) as the nitrogen and carbon sources, respectively. Decomposition of LVFX occurred at elevated temperature aﬀording N , N 0 -desethylene levofloxacin hydrochloride ( N , N 0 -DLH). The crystal structure of the resulting inclusion complex N , N 0 -DLH@Q[6] (cid:2) [CdCl 4 ] 2 (H 3 O) (cid:2) 9H 2 O was determined, where N , N 0 -DLH is protonated on each of the terminal nitrogens and the quinone functionality is a quinol which forms an intramolecular hydrogen bond to the carboxylic acid. The synthesized N , N 0 -DLH containing Q[6]-CDs emitted intense blue fluorescence with high photostability and exhibited stability at high ionic strength. In particular, the original rigid macrocyclic skeletons of these hosts were retained during the fabrication process, which helps in uniquely distinguishing them from other reported CDs. Meanwhile, the performance of the Q[6]-CDs was characterized using fluorescence and NMR spectroscopies. Subsequently, using the obtained Q[6]-CDs, an eﬃcient sensing method for L -tryptophan ( L -Trp) and capecitabine (CAP) has been developed based on macrocyclic host–guest chemistry. Under applicable conditions, the detection limits for L -Trp and CAP were calculated to be 5.13 (cid:3) 10 (cid:4) 8 M and 1


Introduction
Carbon dots (CDs) have attracted extensive attention in recent years because of their excellent optical and chemical properties and have many potential applications. 1,2 CDs were discovered by accident during the processing of single-walled carbon nanotubes (SWCNT). 3 Carbon dots (CDs), as one of the important family members of carbon-based materials, not only have similar physical and chemical properties to those of representative carbon-based materials, but also exhibit low toxicity, photostability, biocompatibility, multiphoton excitation, upconversion photoluminescence and chemiluminescence. 4,5 These excellent characteristics mean that CDs are widely used in the sensing and biomedical fields. [6][7][8] In addition, because of their good biocompatibility and photostability, CDs can be considered as an excellent substitute for more toxic semiconductor quantum dots or unstable organic fluorophores (for biological applications). [9][10][11] With the development of research on CDs, researchers have put forward a number of simple synthetic methods, such as laser ablation, pyrolysis, electrochemical synthesis, supported synthesis, acid oxidation, microwave-assisted synthesis and hydrothermal synthesis. [12][13][14][15][16][17] Among them, CDs synthesized by hydrothermal methods can generate hydroxyl groups, carbonyl groups, carboxyl groups, ether groups, epoxy groups, etc. at the surface. These groups usually have good reactivity, thus affording these nanoparticles excellent reactivity. 18 Therefore, the direct preparation of CDs with adjustable particle size and fluorescence characteristics in water has broad application prospects. Here, cucurbit[n]urils are also shown to be a good raw material for CD synthesis.
Cucurbit[n]urils 19,20 (usually abbreviated as Q[n]s or CB[n]s, n = 5-8, 10, 13-15) are macrocyclic compounds. Generally speaking, Q[n]s possess a rigid cavity and two open ports, with the latter composed of multiple carbonyl oxygens and they have excellent molecular selective binding ability in water. Therefore, cucurbit[n]urils play an important role in supramolecular chemistry and material science. [21][22][23][24] In Q[n]s, the nearly neutral charge distribution on the cavity surface is beneficial to the combination of guest molecules and the formation of host-guest inclusion compounds. 25 36,37 On the other hand, in order to improve the luminescent properties of CDs, the introduction of heteroatoms such as N, B, S, P, Si and Se in the hydrothermal synthesis of CDs has proved to be an effective strategy. [38][39][40][41][42][43] Fluorine is an element with high electronegativity, which can improve the fluorescence properties of CDs. F-doped CDs have the advantages of biocompatibility, cell absorption and stability. 44 However, up to now, there are few research reports on F-doped CDs.
In this paper, carbon dots based on Q [6] were synthesized by using Q [6] and levofloxacin (LVFX) containing C, N, O and F as potential carbon sources. For convenience of description, we refer to these as Q[6]-CDs herein. Q[6]-CDs with high solubility and stability were synthesized by hydrothermal synthesis. The Q[6]-CDs were obtained by simply controlling the fusion and carbonization of LVFX with the aromatic skeleton structure substituted by C, N, O and F with Q [6] in water. Scheme 1 depicts the structural diagram of Q [6] and LVFX.
However, it is worth mentioning that research has shown that LVFX can be oxidized and degraded at high temperature, or by light and other factors during either preparation or storage, and N,N 0 -desethylene levofloxacin hydrochloride (N,N 0 -DLH) is one of the important impurities. 45 In this work, the carbon dots synthesized by LVFX and Q [6] were prepared at 180 1C. Given this, the LVFX present decomposes into N,N 0 -DLH, and the obtained carbon dots result from the combination of N,N 0 -DLH and Q [6]. Furthermore, the single crystal structure of LVFX and Q [6], prepared from a sample synthesized at high temperature, also revealed that the guest molecule LVFX had decomposed. Significantly, it is found that the carbon dots assembled from Q [6] and N,N 0 -DLH possessed excellent fluorescence properties. These results provide insight into the degradation properties of LVFX, as well as a strategy for forming carbon dots incorporating N,N 0 -DLH. Thanks to the advantages of Q[6]-CDs, such as good water solubility, excellent fluorescence, high sensitivity and fast response, the Q[6]-CDs were selected as the fluorescent probe, and 20 essential amino acids and the anti-tumor drug capecitabine (CAP) agent were identified and detected. It was found that the Q[6]-CDs were specific and sensitive for the detection of L-tryptophan (L-Trp) and capecitabine (CAP). At the same time, we also preliminarily studied the possible interaction mechanism of the functionalized Q[6]-CDs with L-Trp and CAP, respectively. On this basis, the application of Q [6]-CDs in the fluorescence detection and identification of L-Trp and CAP is further broadened. This work also lays down the foundation for the detection of L-Trp and CAP in cells, and provides a new application for such CDs.

Experimental
Chemicals and reagents Q [6] was synthesized according to the literature. 46 Levofloxacin, capecitabine and all amino acids were purchased from Aladdin (Shanghai, China). Deionized water was used throughout. All amino acids and capecitabine were prepared with deionized water in a solution of 0.02 M concentration.

H NMR spectral experiments were carried out on a Nippon
Denshi JEOL FT-400 spectrometer. The UV-vis spectra were recorded at room temperature using an 8453 UV-vis spectrophotometer purchased from Agilent (Agilent Technologies, Santa Clara, California, USA). Fluorescence spectra measurements were performed on a Varian Cary Eclipse fluorescence spectrophotometer equipped with a xenon discharge lamp. A model 818 pH meter (Orilon of America) was used to adjust the pH of the solution. Fourier transform infrared (FTIR) spectra were recorded on a Bruker Vertex as KBr pellets. Crystallographic data were collected on a Bruker D8 VENTURE diffractometer using graphite monochromated Mo Ka radiation (l = 0.71073 Å). and the observed chemical shift is reported in parts per million (ppm) relative to the built-in tetramethylsilane (TMS) standard (0.0 ppm).

Fourier transform infrared spectroscopy (FTIR) sample
The above Q[6]-CDs and spectrally pure potassium bromide were dried in a vacuum drying oven and mixed at a ratio of about 1 : 200. The mixture was thoroughly ground and mixed in an agate mortar, and then the tablets were pressed on a pressure machine for sample preparation using the matching mold of an infrared spectrometer. The absorption/transmission spectrum was recorded by an infrared spectrometer with a scanning range of 400-4000 cm À1 .

Preparation of UV-vis solution
10 mg Q[6]-CDs solid was dissolved in 25 mL ultrapure water to obtain a concentration of 400 mg mL À1 reserve solution. Then 5 mL of the reserve solution was diluted to 100 mL with ultrapure water to obtain 20 mg mL À1 Q[6]-CDs solution. 3 mL of the 20 mg mL À1 solution was transferred into a UV cuvette, and the UV-visible absorption spectrum of the Q[6]-CDs was measured by UV-visible spectrophotometer (scanning speed was medium, scanning range was 200-800 nm).

Preparation of fluorescence spectrum (FL) solution
3 mL of 20 mg mL À1 solution was transferred into a fluorescence colorimetric dish, and the fluorescence emission spectrum of the Q[6]-CDs was measured by fluorescence spectrophotometer. The fluorescence emission spectra of the carbon quantum dot solution in each centrifuge tube excited at 245 nm were measured.

Detection of amino acids by Q[6]-CDs
Different amino acids (L-Ser, L-Aal, L-Phe, L-Asn, L-Leu, L-Thr, L-Pro, L-Lys, L-Arg, L-Tyr, L-Cys, L-Gly, L-Ala, L-Iso, L-Gln, L-Asp, L-Met, L-Glu, L-His, L-Trp) were added into the prepared fluorescent probe standard solution (3 mL, 20 mg mL À1 ) for detection (the added amino acid concentration is 0.02 mol L À1 , and the volume is 50 mL). The fluorescence emission spectra of the carbon quantum dot solution in each centrifuge tube excited at 245 nm were measured, and the change of fluorescence intensity was compared with that of blank solution without the amino acids.

Detection of capecitabine by Q[6]-CDs
CAP was added into the prepared fluorescent probe standard solution (3 mL, 20 mg mL À1 ) for detection (the added CAP concentration is 0.02 mol L À1 , and the volume is 50 mL). The fluorescence emission spectra of the carbon quantum dot solution in each centrifuge tube excited at 245 nm were measured, and the change of fluorescence intensity was compared with that of blank solution without CAP.

Synthesis and purification of Q[6]-CDs
LVFX (20 mg, 0.05 mM) and Q [6] (50 mg, 0.05 mM) were dissolved in deionized water (50 mL). The solution was stirred for 60 min and the solution was transferred into a Teflon autoclave. After being heated at 180 1C for 12 h, the autoclave was cooled to room temperature. The reaction solution was centrifuged at 4000 rpm for 45 min, and the supernatant was collected. The resulting supernatant was further filtered through a 0.22 mm filter membrane to remove the large or agglomerated particles. After removing the solvents and freezedrying, a brown powder was obtained (35 mg, yield 50%).

Characterization of Q[6]-CDs
The Q[6]-CDs were synthesized from pure LVFX and Q[6] using a conventional hydrothermal route (for details, see the Experimental section). In the UV/vis spectra, the Q[6]-CDs exhibited maximum adsorption peaks at 275 nm in an aqueous solution (Fig. 1a). The absorption peak at 275 nm was attributed to the pp* transitions of the CQC bonds. Interestingly, the solution of Q[6]-CDs emitted a blue fluorescence (l max = 430 nm) (Fig. 1b). No UV-vis adsorption and emission behaviour was observed in the aqueous solutions of pristine Q [6].
In order to better understand the role of Q [6] in the formation of Q[6]-CDs, the host-guest interaction between LVFX and Q [6] was studied before the hydrothermal reaction. First, a pH titration of LVFX and the LVFX@Q [6] complex over the range pH 2-12.0 was performed using UV/vis spectra, as shown in Fig. S1 (ESI †). For the free LVFX and LVFX@Q [6], minimal changes in the UV-vis absorption spectra were observed at pH r 5 and pH Z 7, but there was a significant change in the absorption spectra at l = 288 nm between pH 5.0 and 7.0. To study the binding interaction of LVFX with Q [6], subsequent investigations were carried out on a solution of pH 7.0. Subsequently, according to the 1 H NMR spectra of Q [6] and LVFX, the interaction between Q[6] and LVFX was studied. As shown in Fig. S2, (ESI †) after the addition of 1.0 equiv. of Q [6], H i , H j , H a , H b , H c , H d , H e , H g and H f the proton peaks corresponding to LVFX were shifted downfield, and the groups of the LVFX guest are located at the portals of the host. Meanwhile, the 1 H NMR spectra of N,N 0 -DLH@Q [6], LVFX@Q [6] and Q[6]-CDs were compared (Fig. S3, ESI †). It is evident that the Q[6]-CDs contain N,N 0 -DLH, which is the result of the oxidative decomposition of LVFX following exposure to high temperature. We then carried out a series of studies on these Q[6]-CDs.
At the same time, the fluorescence spectrum of free LVFX indicated that the organic precursor exhibited a greenish blue emission with the maximum wavelength at around 470 nm at room temperature in water, and the UV-vis absorption and fluorescence emission changes were observed upon the addition of the Q [6] host to the LVFX solution with a 1 : 1 binding fashion (host/guest, mole ratio) (Fig. S4, ESI †), which further supported the portal binding model of the Q[n]s. Importantly, the original proton peaks of Q [6] were observed in the 1 H NMR spectrum of the Q[6]-CDs (Fig. S5, ESI †), which indicated that the rigid macrocyclic skeleton of the Q [6] remained intact during the hydrothermal reaction with LVFX.
Then, we studied the effect of pH on the fluorescence properties of the Q[6]-CDs (Fig. 1c). Research has found that the fluorescent emission intensity of the Q[6]-CDs rises to the largest value at pH 5.5, with the solution pH changing from 2 to 5.5, and then decreases as the pH changes from 5.5 to 12.0. This shows that the Q[6]-CDs are very sensitive to pH. Such behaviour is similar to CDs containing hydroxyl and carboxyl moieties. 47 In addition, to better understand the difference between the groups LVFX@Q [6] and Q[6]-CDs, the FT-IR spectra were analyzed to identify surface functional groups of LVFX@Q [6] ( Fig. S6 and Table S1, ESI †). The strong absorption peaks at 1774 and 1734 cm À1 are assigned to the stretching vibrations of CQN and CQO, respectively. 48,49 The sharp peaks at 1684 cm À1 and 1490 cm À1 are in accord with the CQO stretching and N-H bending. 50 However, the FT-IR spectrum also confirmed that the Q[6]-CDs contains functional groups of ÀOH (3092 cm À1 ) and CQO (1705 cm À1 ), 51 and -C-N (1400 cm À1 ), -C-O and -C-O-C (1000-1200 cm À1 ), 52 -COOH (1050 cm À1 ) were present. New absorption bands belonging to the stretching vibration of -C-H (2867 cm À1 , 2951 cm À1 ) in the Q[6]-CDs, 53 and the stretching vibrations of C-F bond at 1249 cm À1 were observed 54 (Fig. 1d). Furthermore, the structure and morphology of the Q[6]-CDs were characterized by transmission electron microscope (Fig. S7, ESI †) and X-ray powder diffraction (Fig. S8, ESI †).
Also, the chemical composition of the Q[6]-CDs was determined by X-ray photoelectron spectroscopy. Fig. 2a Fig. 2b shows the fine spectrum of C1s, and the absorptions at 286.18 eV, and 289.18 eV, which indicate the existence of C-C/CQC, and CQO bonds, respectively. In the N1s spectra (Fig. S9, ESI †), absorptions at 400.38 eV can be attributed to C-N bonds. In the case of the O1s spectra (Fig. 2c), absorptions at 532.38 eV correspond to CQO and C-OH bonds. In the F1s spectra (Fig. 2d)  Description of the crystal structure of N,N 0 -DLH@Q [6] The X-ray crystal structure analysis provided unequivocal proof of the 1 : 1 binary complex assembly (Fig. 3). The crystal structure reveals that hydrogen bonding is the dominant interaction driving the formation of N,N 0 -DLH@Q [6]Á[CdCl 4 ] 2 (H 3 O)Á 9H 2 O (Fig. 3). Table S2 (ESI †) Fig. 3. The protonation of the guest helps to facilitate two hydrogen bonds between N25 and the Q6 as shown below. Note the N,N 0 -DLH is thrice protonated at the each of the terminal nitrogens and the quinone functionality is a quinol; the latter forms an intramolecular hydrogen bond to the carboxylic acid. Interestingly, the guest has racemised during the synthesis, i.e. it is no longer racemically pure at the ethylene bridge attached to the ring system.
There is minor disorder in the positions of the unbound water molecules and some are split over two positions. It was not possible to locate hydrogen atoms on these. Similarly, it was not possible to identify the location of the single H 3 O + ion. There is good evidence for the protonation of the guest molecule. Electron density consistent with the presence of hydrogen atoms is found on the atoms N25, N26, and O13 suggesting that these atoms are protonated. Similarly, the bond C41-N26 is too short to be a single bond and the bond C46-O13 is too long to be a double bond; the hydrogen attached to O13 also forms an intramolecular hydrogen bond to O14. For a view of the asymmetric unit and the guest see Fig S13 and S14 (ESI †).

Fluorescence detection of the Q[6]-CDs towards L-tryptophan
For practical detection, a favorable fluorescent probe should have high selectivity to the analyte. Herein, the fluorescence response of Q[6]-CDs to a series of common amino acids was  determined. When L-Trp was added to an aqueous solution of Q[6]-CDs, the fluorescence intensity at 430 nm was strongly reduced (Fig. 4), and was accompanied by an obvious blue shift of 52 nm, while the addition of the other 19 amino acids did not cause any significant fluorescence changes for the Q[6]-CDs. This demonstrated the potential of the prepared Q[6]-CDs for the detection of L-Trp, and except for L-Trp, the remaining 19 amino acids had no significant effect on the fluorescence intensity at 430 nm (Fig. 5).
To further understand the recognition capability of the Q[6]-CDs toward L-Trp, luminescence titration experiments were performed. Fig. 6a shows that the fluorescence intensity of the Q[6]-CDs decreases gradually, and the fluorescence emission of the Q[6]-CDs was tunable from 430 to 378 nm with the continuous addition of L-Trp. The increasing intensity was accompanied by a clearly enhanced UV-vis absorption intensity upon the addition of an increasing concentration of L-Trp to an aqueous solution of the Q[6]-CDs (Fig. S15, ESI †). In addition, the fluorescence intensity observed at 430 nm exhibited a good linear relationship with the L-Trp concentration over the range of 0.75-1.8 mM (Fig. 6b), and the LOD was calculated to be 5.13 Â 10 À8 M. This indicated that the Q[6]-CDs were very sensitive toward the detection of L-Trp in aqueous media. To evaluate the selectivity of the Q[6]-CDs towards L-Trp detection, the effects of the co-existing amino acids were investigated under the same conditions. As shown in  Similar NMR spectral changes were observed in the LVFX and L-Trp systems. As the concentration of LVFX was increased to B1.0 eq., for the L-Trp guests, the signals of all the guest protons move upfield by different degrees (Fig. S19, ESI †). Therefore, the results show that the possible mechanism of Q[6]-CDs fluorescence quenching caused by the addition of L-Trp is that the indole part of L-Trp is a planar aromatic ring structure. At the same time, the main chromophores (conjugated p domain and molecular fluorophore) in the Q[6]-CDs are  also planar aromatic ring structures. Due to their strong intermolecular p-p stacking interaction, their large planar polycyclic aromatic structure promotes them to stack in a directional way, which will lead to the nonradiative transition of the excited state back to the ground state, resulting in fluorescence quenching. 55

Analytical performance of Q[6]-CDs towards capecitabine
Capecitabine can prolong the survival time of various cancer patients, and is widely used in the treatment of advanced primary or metastatic breast cancer, rectal cancer, colon cancer and gastric cancer. Therefore, it is very important to choose a sensor to identify and detect CAP. With this in mind, we have probed the ability of the Q[6]-CDs to detect CAP. As shown in Fig. 7, when CAP is added to the Q[6]-CDs solution, the fluorescence intensity is quenched at 430 nm, and the emission wavelength is blue shifted from 430 nm to 396 nm. Furthermore, with the gradual dropping of CAP into the Q[6]-CDs solution, the UV-vis absorption intensity of the system gradually increases (Fig. S20, ESI †). The mechanism of the fluorescence quenching caused by the addition of CAP to Q[6]-CDs was next investigated. Firstly, the 1 H NMR spectrum (Fig. S21, ESI †) of the CAP titration Q [6] was analyzed. It can be seen from the figure that when the molar ratio of CAP to Q[6] is 1 : 1, only the alkyl chain (H a , H b , H c , H d ) in the CAP molecule shifts upfield, and the rest shift downfield, which indicates that only the alkyl chain of the CAP extends into the cavity of the Q [6]. In addition, the interaction between CAP and Q[6]-CDs was further analyzed by 1 H NMR spectroscopy (Fig. S22, ESI †), and it was found that all protons of the CAP molecule shift upfield. In order to explain this phenomenon, the NMR spectrum of the interaction between CAP and LVFX was analyzed (Fig. S23, ESI †). It was found that when the same amount of CAP was dripped into LVFX, all the protons of the CAP molecules also shifted upfield. Therefore, the reason of fluorescence quenching caused by the addition of CAP to Q[6]-CDs is similar to the above-mentioned L-Trp system, and it may also be due to the planar aromatic ring structure of the CAP. There is p-p stacking between the planar aromatic ring structure of CAP and the planar aromatic ring structure in Q[6]-CDs, and the polycyclic aromatic structure between them forces them to stack in a directional way. This accumulation will cause the excited state to transition back to the ground state by nonradiative pathway, resulting in fluorescence quenching.

Conclusions
In this paper, Q [6] and LVFX were used as carbon sources, and Q[6]-CDs were synthesized by simply controlling the fusion and carbonization of LVFX with Q [6]. At the same time, it was found that there is N,N 0 -DLH in the Q[6]-CDs, which is due to the high temperature degradation of LVFX to N,N 0 -DLH, whereby the piperazine ring of LVFX has decomposed into an ethylenediamine group. The Q[6]-CDs have the advantage of high sensitivity, fast response, good selectivity, high water solubility and excellent fluorescence performance. The Q[6]-CDs, as a new water-soluble nano-sensor, can efficiently and simply detect L-Trp and CAP. The detection limits of Q[6]-CDs for L-Trp and  CAP are 5.13 Â 10 À8 M and 1.48 Â 10 À8 M, respectively. In addition, the possible mechanism of detecting L-Trp and CAP by Q[6]-CDs was analyzed, which may be due to the fact that both L-Trp and CAP have planar aromatic ring structures, and there is also a planar aromatic ring structure in the Q[6]-CDs. There is p-p stacking between the planar aromatic ring structures of L-Trp and CAP and the planar aromatic ring structures in Q[6]-CDs, respectively, and their large planar polycyclic aromatic structures force them to stack in a directional manner. This kind of accumulation will cause the excited state to transition back to the ground state by a non-radiative pathway, thus causing fluorescence quenching. This new type of fluorescence sensor has feasibility and potential application prospects in future fluorescence detection and identification.

Conflicts of interest
The authors declare no competing financial interest.