Buhong
Gao
a,
Yu
Sun
b,
Yingchun
Miao
a,
Li
Xu
*b and
Zhongxia
Wang
*c
aAdvanced Analysis & Testing Center, Nanjing Forestry University, Nanjing 210037, China. E-mail: gaobuhong@126.com
bCollege of Science, Nanjing Forestry University, Nanjing, 210037, China
cSchool of Chemistry and Chemical Engineering, Yancheng Institute of Technology, Yancheng 224051, China. E-mail: wangzx198411@163.com
First published on 6th November 2019
Highly luminescent nitrogen and phosphorus co-doped graphene-analogous flakes (NPCGFs) were synthesized by a one-pot simple hydrothermal reaction using β-cyclodextrin (β-CD), vinylphosphoric acid (VPA), and o-phenylenediamine (oPD) as the precursors. VPA, as an important organic P-containing monomer, was selected as the phosphorus source to generate additional conjugated and effective binding sites on the surface of the NPCGFs. This synthetic strategy not only allows enhancement of structural rigidity, but also effectively eliminates surface traps of the NPCGFs, resulting in an improved fluorescence quantum yield (FL QY) of the NPCGFs. Additionally, oPD simultaneously acts as a nitrogen source and enables amino functionalisation of the NPCGF surface in the synthesis process. The NPCGFs (QY, 32.49%) are irregularly shaped with a typical diameter of approximately 54 nm and display strong fluorescence, with excitation/emission maxima of 360/445 nm. It was found that the NPCGFs can serve as a multifunctional FL probe for pH measurement and quercetin (Qc) detection. A linear relationship exists between the decrease in FL intensity and the concentration of Qc in the range from 0.35 to 30 μg mL−1 as well as the pH variation between 4.0 and 7.0. The probe was further applied to the determination of Qc in living cells.
The P atom has been widely used for the preparation of P-doped CNMs because it has properties comparable to those of carbon atoms and also possesses multivalent electrons for bonding with carbon. For example, Yang et al. synthesized P-doped carbon dots with tunable emission using hydrothermal treatment of glucose in the presence of monopotassium phosphate.5 Xu et al. prepared N and P co-doped high-FL carbon dots using sodium citrate and diammonium phosphate as raw materials.6 Wang et al. synthesized N and P co-doped carbon polymers by a hydrothermal approach using urea and H3PO4 as the C–N and P sources, respectively.7 Recent research involving the P-doping of CNMs mainly with P from inorganic sources aroused our interest to further examine the corresponding performance of its counterpart by P-doping CNMs with P from organic sources. Thus far, research on the P-doping of CNMs using organic P sources is still rare. Hence, it is worthwhile to prepare new P-doped CNMs with high quantum yields and selectivity using different organic P monomers.
As the most active antioxidant of the flavonol family, quercetin (Qc) is widely present in vegetables, flowers, fruits, and especially in traditional Chinese herbs.8–11 In the past few years, there has been great interest in Qc because of its antioxidant properties affecting human DNA in vitro,12 and also for its antitumour and antiviral properties, and its ability to aid in adjusting the immune system.13,14 As a consequence, developing an effective analytical technique for selective and sensitive detection of trace amounts of Qc is essential in biochemistry, clinical medicine, and natural pharmaceutical chemistry. Thus far, a series of methods have been developed to detect Qc, including high-performance liquid chromatography,15 electrochemistry,16 and Raman spectroscopy.17 However, each of these methods is limited by severe disadvantages, such as time-consuming processes, complicated equipment, and high costs, which severely limit their wider applications. In contrast, FL analysis can provide a non-destructive, ultrasensitive, and simple detection platform that is free from complicated pretreatment processes. To date, some materials including gold nanoclusters,18 organosilane-functionalized carbon dots,19 and carbon nanoribbon polymers20 have been used as FL sensors for Qc detection. Despite these good examples, it is still very necessary to explore novel FL probes with low toxicity or that are non-toxic, and that also possess ease of synthesis with high photostability and quantum yield (QY) for sensitive and selective determination of Qc.
In the current study, as a novel method for doping CNMs, low-cost N and P co-doped fluorescent graphene-analogous flake (NPCGF) nanoprobes with excellent optical properties, high stability, and superior water dispersibility were successfully synthesized for the first time by hydrothermal treatment of β-cyclodextrin, vinylphosphoric acid, and o-phenylenediamine with high QY of approximately 32.49%. Surprisingly, the NPCGFs showed highly efficient FL quenching ability in the presence of Qc and H+ cation, indicating their potential application as a dual-readout assay platform. The principle of the proposed Qc and H+ cation-sensing concept is shown in Scheme 1. As far as we know, this is the first example of the construction of a FL assay platform for Qc and pH based on highly efficient NPCGFs. Additionally, NPCGFs with excellent selectivity and satisfactory recovery were further used to detect Qc in complex real samples and living cells.
In order to understand the mechanism governing the formation of the NPCGFs, control experiments were conducted, and the products were tested by FL spectra. Based on our previous work, we revealed that the CNMs from pure β-CD emitted nearly no fluorescence. The FL intensity was slightly increased by analysing both comonomers of β-CD and VPA (Fig. S1,† blue line). Interestingly, the FL intensity of NPCGFs at 445 nm clearly increases because of the comonomers of β-CD, VPA, and oPD (Fig. S1,† black line). In order to further reveal the role of oPD, two phenylenediamines isomers, (m-phenylenediamine (mPD) and p-phenylenediamine (pPD), were replaced with oPD to prepare CNMs under the same reaction conditions as those of NPCGFs.
It was found that the synthesized CNMs from mPD and pPD exhibited weak emission at 445 nm compared to the NPCGFs. Moreover, the maximum emission peaks of the products from pPD and mPD were 522 nm with excitation of 440 nm (Fig. S1,† green line), and 536 nm with the excitation of 380 nm (Fig. S1,† red line), respectively. Subsequently, phenylenediamine analogue (o-dihydroxybenzene) was also used to study the effect of chemical composition on the FL of CNMs. The obtained spectrum was significantly different from that of the NPCGFs (Fig. S1,† purple line), which could be due to the synergetic effect of the simultaneous existence of comonomers of β-CD, VPA, and oPD for prepared NPCGFs. The chemical reaction between the different active sites during the synthesis process may have resulted in the different unique arrangement of molecular structure, and ultimately caused the difference in the optical property.
We further verified whether CC bonds in VPA affected the FL feature of the CNMs. Compared with the NPCGFs, the CNMs obtained from phosphoric acid as the phosphorous source have a relative low FL intensity (Fig. S1,† gray line). The reason for this is because the introduction of CC bonds in VPA increased the number of active sites during the reaction, resulting in a stronger structural rigidity of the NPCGFs. All the above results suggested that the relative positions and chemical composition of substituent groups were the principal factors in tuning the FL properties of the CNMs.
Finally, the mass ratio between β-CD and oPD was investigated in parallel reactions, and a ratio of 1:1 was found to be optimal for the strongest FL (Table S2†). The TEM images (Fig. 1a and b) clearly reveal that the NPCGFs are graphene-analogous structures with irregular shapes and a size distribution between 40 and 70 nm; the average size is approximately 54 nm, which was calculated by measuring several hundred particles. In addition, these nanoparticles are homogeneously dispersed without apparent aggregation. The SAED pattern (Fig. 1c) clearly shows no well-resolved lattice fringes, indicating the amorphous nature of the NPCGFs.23 As shown in Fig. 1d, the EDX spectrum shows four peaks corresponding to C, N, O, and P, respectively. The AFM image (Fig. 1e) reveals a typical topographic height of 0.5–1.5 nm (Fig. 1f), as derived from the topographical height profile (section analysis), demonstrating that the construction of some of the NPCGFs may include a multi-layered structure.
Fig. 1 (a, b) TEM images with different magnifications, (c) SAED pattern, (d) EDS spectrum, (e) AFM image, and (f) height distributions of the NPCGFs. |
The XRD profile (Fig. 2a) exhibits a broad peak at 2θ = 23.96°, corresponding to a disordered graphitic structure.24 The Raman spectrum (Fig. 2b) contains characteristically wide D and G bands around 1360 and 1590 cm−1, which are typical for amorphous carbons or disordered graphite, respectively. The intensity ratio of the D and G bands is 1.35, indicating the very low degree of graphitization of the resulting hydrothermal carbon material,4 which further demonstrates that the NPCGFs obtained via the hydrothermal carbonization of β-CD in the presence of VPA and oPD are amorphous. This finding is in agreement with the SAED pattern and XRD analysis.
XPS and FT-IR spectroscopy were further performed to study the functional groups and the chemical composition of the NPCGFs. The survey spectrum (Fig. 3a) displays four strong signals from C 1s at 285 eV, O 1s at 531 eV, N 1s at 400 eV, and P 2p at 133 eV, with atomic percentages of 60.53%, 20.35%, 10.49%, and 8.63%, respectively. The high-resolution of C 1s (Fig. 3b) can be well deconvoluted into five surface composites, corresponding to CC at a binding energy of 284.09 eV, C–C at 284.83 eV, C–N/C–P at 285.87 eV, C–O at 286.73 eV, and CO at 287.39 eV.6 The O 1s spectrum (Fig. 3c) shows three peaks at 531.56 eV, 532.17 eV, and 533.59 eV, which were attributed to CO, C–OH, and C–O–C groups, respectively.25Fig. 3d shows the XPS spectrum of N 1s peaks at 399.10 eV and 400.64 eV, which were attributed to pyrrolic-like N and N–C/N–H, respectively.26 As shown in Fig. 3e, the high resolution of P2p exhibits three peaks at 132.11 eV, 132.84 eV, and 133.75 eV, which were associated with the P–O, PO, and P–C/P–N binding energies, respectively.27
Fig. 3 The (a) XPS survey showing the (b) C 1s, (c) O 1s, (d) N 1s, and (e) P 2p spectra of the prepared NPCGFs. |
The FT-IR spectrum also exhibits the characteristic absorption bands of the stretching vibration of PO at 1205 cm−1 and P–O at 1021 cm−1 (Fig. 2c), which indicated that the functional monomer (VPA) was successfully incorporated into the NPCGFs.7 The peak at 1635 cm−1 was associated with vibrational absorption of CC and CO, and the band at 1454 cm−1 refers to the C–N bond.28 The peaks at 1385 cm−1 and 1153 cm−1 were attributed to the C–O stretching vibration and C–O–C stretching vibration.27 The absorption bands of O–H and N–H appeared at 3437 cm−1 and 1580 cm−1, respectively. All these absorption peaks indicate that the NPCGFs were rich in hydroxyl, amine, and phosphate groups on the framework. The above data, including the XPS and FT-IR results, indicated the successful incorporation of P and N into the NPCGF framework.
The UV-Vis absorption and FL spectra were characterized to further explore the optical properties of the prepared NPCGFs. The UV-Vis absorption spectrum of the NPCGFs (Fig. 4a, curve a) shows several absorption maxima/bands at approximately 240 nm, 266 nm, 273 nm, 315 nm, 360 nm, and 425 nm. The peaks at 240 nm and 266 nm corresponded to the π–π* transition of the aromatic CC bond and n–π transition of C–N, while the peak at 273 nm was attributed to the n–π* transition of the aromatic π system CO, and all these do not typically generate FL.29 The absorption bands at 315 nm, 360 nm, and 425 nm, in the lower-energy region, could be attributed to the trapping of the excited state energy by the surface states, which results in strong fluorescence.30,31 Furthermore, the maximum excitation and emission peaks of the NPCGFs were located at 360 nm and 445 nm (Fig. 4a, curves b and c), respectively, with a stock shift of 85 nm. Similar to the characteristics of CNMs, NPCGFs also exhibited an excitation-dependent emission (Fig. 4b), indicating the multicolour properties of the NPCGFs. The above feature might be attributed to different surface states and sizes of the NPCGFs.27 The absolute quantum yield (QY) of the NPCGFs was 32.49% using water as the solvent (Fig. S2†).
FL0/FL = 1 + Kc, | (1) |
Until now, many methods have been adopted to detect Qc that show excellent sensitivity and a low limit of detection (LOD), but there are still some limitations consisting of a time-consuming process, multi-step sample preparation, toxic drugs, and/or specialised skills. Fortunately, the NPCGF probe has the advantage of being non-toxic to the environment, and the fabricated sensor also satisfies the requirement of one site and excellent selectivity (Table S4†).
To verify this conclusion, the zeta potential was obtained. The zeta potential of the NPCGFs was +8.15 mV (Fig. S5†), which further confirmed the possibility of forming a polymer between Qc and the NPCGFs through electrostatic interactions, resulting in electron or energy transfer and causing FL quenching of the NPCGFs. Thus, Qc was slowly added to the NPCGFs under the excitation state, and the electron or energy, which was transferred in the Qc by the NPCGFs, was back-donated in the electron-deficient NPCGFs, leading to strong electronic interaction34 and thereby decreasing its luminescence intensity.
To further reveal the mechanism of FL quenching, time-resolved FL spectra were measured. As shown in Fig. S6,† the FL spectra of NPCGFs and the mixture of NPCGFs and Qc obey dual exponential decay kinetics, and two lifetimes were acquired, which may be attributed to the differences in the distribution of complex luminescent pathways resulting from multiple unique NPCGFs and/or sites. The average lifetime of NPCGFs in the absence and presence of Qc was approximately 8.72 ns and 7.74 ns, respectively. The relative changes in the FL lifetime of the NPCGFs can be attributed to the increase in the radiative decay rates, indicating that the formation of an extra shell partly through combination with Qc changes the surface states. Additionally, the nonlinear Stern–Volmer plot (Fig. 5d) indicates that the decrease in lifetime of the NPCGFs by Qc obeys a complex quenching mechanism. That is, the NPCGFs, like positively charged cations, coordinate to the negative electron source of the Qc and should act as an electron-deficient compound to bind electron-enriched Qc, and subsequently quench the FL of NPCGFs in the presence of Qc, which might be attributed to an electron- or energy-transfer mechanism.
The property of being highly selective enables accurate detection of Qc in water samples. Water collected from the Xuan Wu River was selected as a real sample to reveal the applicability of NPCGFs for Qc detection, and the standard addition method was used. The collected samples were filtered through a 0.22 mm membrane and then centrifuged at 8000 rpm for 15 min. As listed in Table S5,† the recoveries of Qc from samples to which Qc was added were in the range of 98.90–100.18% with RSD of 3.95–4.82%, indicating that the NPCGF probe is reliable for the detection of Qc in real water samples.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9an02077b |
This journal is © The Royal Society of Chemistry 2020 |