N-Doped Graphene Quantum Dots-Decorated V2O5

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Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

N‑Doped Graphene Quantum Dots-Decorated V2O5 Nanosheet for Fluorescence Turn Off−On Detection of Cysteine Akhilesh Babu Ganganboina,† Ankan Dutta Chowdhury,‡ and Ruey-an Doong*,†,‡ †

Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, 101 Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan ‡ Institute of Environmental Engineering, National Chiao Tung University, 1001 University Road, Hsinchu 30010, Taiwan S Supporting Information *

ABSTRACT: The development of a fast-response sensing technique for detection of cysteine can provide an analytical platform for prescreening of disease. Herein, we have developed a fluorescence turn off−on fluorescence sensing platform by combining nitrogen-doped graphene quantum dots (NGQDs) with V2O5 nanosheets for the sensitive and selective detection of cysteine in human serum samples. V2O5 nanosheets with 2−4 layers are successfully synthesized via a simple and scalable liquid exfoliation method and then deposited with 2−8 nm of N-GQDs as the fluorescence turn off−on nanoprobe for effective detection of cysteine in human serum samples. The V2O5 nanosheets serve as both fluorescence quencher and cysteine recognizer in the sensing platform. The fluorescence intensity of N-GQDs with quantum yield of 0.34 can be quenched after attachment onto V2O5 nanosheets. The addition of cysteine triggers the reduction of V2O5 to V4+ as well as the release of N-GQDs within 4 min, resulting in the recovery of fluorescence intensity for the turn off−on detection of cysteine. The sensing platform exhibits a two-stage linear response to cysteine in the concentration range of 0.1−15 and 15−125 μM at pH 6.5, and the limit of detection is 50 nM. The fluorescence response of N-GQD@V2O5 exhibits high selectivity toward cysteine over other 22 electrolytes and biomolecules. Moreover, this promising platform is successfully applied in detection of cysteine in human serum samples with excellent recovery of (95 ± 3.8) − (108 ± 2.4)%. These results clearly demonstrate a newly developed redox reaction-based nanosensing platform using NGQD@V2O5 nanocomposites as the sensing probe for cysteine-associated disease monitoring and diagnosis in biomedical applications, which can open an avenue for the development of high performance and robust sensing probes to detect organic metabolites. KEYWORDS: N-GQDs, V2O5 nanosheet, turn off−on sensing, cysteine detection, redox reaction, fluorescence

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INTRODUCTION Cysteine, one of the essential amino acids containing thiol group, plays an important role in cellular processes like intracellular redox homeostasis through the equilibrium between free thiols and oxidized disulfides.1 Cysteine also provides a modality for the intermolecular cross-linking of proteins to maintain the enzymatic activity in human body. Usually the level of cysteine, homocysteine, and glutathione in the plasma is highly correlated with pathophysiology of certain human diseases, such as slow growth retardation, acquired immunodeficiency syndrome, Alzheimer’s, and Parkinson’s diseases.2 Thus, the search of sensitive and selective fluorescent sensors for detection and discrimination of these biothiols has stimulated intense interest. Recent studies have provided a wide variety of techniques for the determination of cysteine including liquid chromatography, flow injection, voltammetry, and capillary zone electrophoresis.3,4 However, these techniques need expensive instruments, skilled persons and tedious procedures for sample preparation, which limit their application. Hence, there is © XXXX American Chemical Society

always a thirst among the scientists to develop cost effective and facile sensing methods for rapid detection of cysteine.5 Several studies have developed electrochemical sensors for the detection of cysteine. Selvarajan et al. have used the functionalized BaTiO3 nanoparticle film-based self-powered biosensor for the detection of cysteine.6 Geng et al. have developed molybdenum nitride/nitrogen-doped multiwalled carbon nanotube hybrid for electrochemical detection of cysteine.7 Although these methods can reach a low detection limit, the electrochemical sensors are difficult in acquiring the required specificity for serum application because of the obvious matrix interference. More recently, fluorescence assay has evolved as one of the most promising analytical methods for the detection of thiol-containing biological molecules due to its significant advantages, such as low cost, simplicity, rapid detection, and real-time imaging.8 The detection principle of Received: October 5, 2017 Accepted: December 11, 2017 Published: December 11, 2017 A

DOI: 10.1021/acsami.7b15120 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Scheme 1. Schematic Illustration of N-GQD@V2O5 Nanocomposite Preparation and Mechanism for Cysteine Detection

fluorescent sensor for determination of cysteine has not been reported. Herein, we have developed a fluorescence turn off−on NGQD@V2O5 sensing platform for the sensitive and selective detection of cysteine in human serum samples. As shown in Scheme 1, the fluorescence quenching of N-GQDs is induced by the electron transfer between N-GQDs and V2O5 when NGQDs are adsorbed onto ultrathin V2O5 nanosheets. The role of V2O5 nanosheets in the sensing platform is to serve as both fluorescence nanoquencher and cysteine recognizer. The fluorescence of N-GQDs is quenched when adsorbed on the surface of V2O5 nanosheets. Addition of cysteine triggers the decomposition of V2O5 nanosheets to V4+, resulting in the release of N-GQDs and the recovery of fluorescence intensity. Furthermore, the introduction of exfoliated V2O5 nanosheets enhances the sensitivity and specificity on cysteine detection at pH 6.5. This new class of fluorescence turn off−on sensing platform displays a sensitive and fast response to cysteine in the linear range of 0.1−15 μM, with a limit of detection (LOD) of 50 nM within 4 min. In addition, the N-GQD@ V2O5 can detect cysteine in human serum samples with excellent recoveries of 95−108%. The fluorescence turn off− on N-GQD@V2O5 sensor developed in this study exhibits superior specificity on cysteine detection over other electrolytes and biomolecules, which clearly demonstrates the promising potential of using N-GQD@V2O5 for cysteineassociated disease monitoring and diagnosis in biological applications.

these sensors is based on the interaction between analytes and probe containing nucleophilic thiol group via cyclization with aldehyde, Michael addition, or cleavage of disulfide, sulfonate esters, and sulfonamide.9 More recently, the fluorescent nitrogen-doped graphene quantum dots (N-GQDs) have emerged as a new class of photostable luminescent nanomaterials with extraordinary optical and electrical characteristics because of their pronounced quantum confinement and edge effects.10 Different from the toxic inorganic quantum dots like CdSe, N-GQDs have the improved properties of relatively good biocompatibility, chemical stability, and low cost,11 which have attracted much attention in a wide range of applications, such as fluorescence sensors,12−17 bio-imaging,18−20 and drug delivery.21 The introduction of fluorescent components and functional nanomaterials has been reported to enhance the selectivity and sensitivity of fluorescent sensors toward analyte detection.22 Two-dimensional (2-D) MnO2 and MoS2 nanosheets can react with GQDs through surface functional groups or surface adsorption to quench the fluorescence intensity of GQDs.23,24 The layered vanadium pentoxide (V2O5) nanomaterial has the excellent properties such as redox activity, wide optical band gap, and good chemical and thermal stabilities, which make this material applicable in sensing devices.25,26 Moreover, the fluorescence of luminescent materials can be quenched by V2O5 when the luminescent substances are on the surface of V2O5 nanosheet. Celestino-Santos et al. have reported the interaction of cysteine with V2O5 for optical detection of cysteine and found that V2O5 is reduced to V4+ in the presence of cysteine.27 It is interesting to note that the N-GQDs have adsorption affinity toward V2O5 surface. The attachment of NGQDs onto V2O5 surface is mainly attributed to the induction of electrostatic interaction between electrophilic V2O5 and nucleophilic N-GQDs. This would further enhance the adsorption of N-GQDs onto the layered V2O5 surface and results in the fluorescence quenching of N-GQDs.28 This gives a great impetus to develop a sensitive turn off−on fluorescent sensor by introducing N-GQDs to V2O5 nanosheet materials in the presence of cysteine. However, the combination of NGQDs with V2O5 as the turn off−on fluorescent sensor has received less attention and the application of N-GQD@V2O5

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EXPERIMENTAL SECTION

Chemicals. Vanadium pentoxide (V2O5), citric acid, urea, sodium dihydrogen phosphate, disodium hydrogen phosphate, and L-cysteine were purchased from Sigma-Aldrich, N,N-dimethyl formamide (DMF) was purchased from Acros Organics. All other reagents were of analytical grade and were used as received without further purification. Solutions used in this study were prepared using bidistilled deionized water purified through a UV-treated Rephile water system (18.2 MΩ cm) unless otherwise mentioned. Exfoliation of Bulk V2O5. Ultrasonic method with DMF as the exfoliating agent was used to prepare V2O5 nanosheets. Hundred milligrams of bulk V2O5 powder was added to 200 mL of DMF solution, and the mixture was shaken overnight at room temperature to suspend the V2O5 powder. The resultant suspension was then sonicated at room temperature for 3 days. After ultrasonic treatment, B


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Figure 1. (a) TEM image, (b) particle size distribution, (c) fluorescence emission spectra at different excitation wavelengths of 310−410 nm, and (d) XPS survey spectrum with C 1s deconvoluted spectrum of as-prepared N-GQDs. The insets of figure (a) and (c) are HRTEM image and fluorescence image irradiated at 360 nm, respectively. the mixture was allowed to sediment and the supernatant was separated. The collected supernatant comprised of exfoliated V2O5 and DMF, which was centrifuged again to harvest exfoliated V2O5 from DMF. The obtained V2O5 nanosheet was washed several times with ethanol to remove residual DMF and then dried in a vacuum oven at 70 °C overnight for further characterization. Preparation of N-GQDs and GQDs. The N-GQDs were prepared by a hydrothermal method using our previously reported method.29 In brief, 0.21 g of citric acid and 0.18 g of urea were added to 5 mL of bidistilled deionized water and stirred to form a clear solution. The solution was then transferred into a 20 mL of Teflonlined stainless steel autoclave tube and heated up to 160 °C for 4 h. The obtained product was collected by adding ethanol into the solution and centrifuged at 5000 rpm for 5 min. The solid was then dispersed in water and dialyzed in a 1 kDa dialysis bag for 24 h, in which the dialysate was replaced for every 8 h, to remove the unreacted reactants. The harvested solution was preserved in the dark at 4 °C for further use. The quantum yield of N-GQDs was determined by the fluorescence method using fluorescein as a standard fluorophore (Φ = 0.79).30 In addition, the as-prepared GQDs were also fabricated for comparison by using the same preparation procedures mentioned above except the addition of urea.29 Characterization of N-GQD@V2O5. The morphology as well as size of nanomaterials, including that of as-prepared V2O5 nanosheets, N-GQDs, and N-GQD@V2O5, were characterized using JEOL JEMARM200F transmission electron microscope (TEM); JEOL JEM2010 high-resolution transmission electron microscope (HRTEM) at 300 kV and Bruker Dimension Edge atomic force microscope (AFM) were used. X-ray diffraction (XRD) patterns were recorded using

Bruker D8 X-ray diffractometer with Ni-filtered Cu Kα radiation (λ = 1.5406 Å) and X-ray photoelectron spectroscopy (XPS) was performed with an ESCA Ulvac-PHI 1600 photoelectron spectrometer from physical electronics using Al Kα radiation photon energy at 1486.6 ± 0.2 eV. Fluorescence spectra of N-GQD-based composites were recorded by Hitachi F-7000 fluorescence spectrophotometer. Detection of Cysteine. The detection of cysteine using NGQD@V2O5 was performed at room temperature in 10 mM phosphate-buffered saline (PBS) buffer solution at pH 5.5−8.0. For cysteine detection, 2 mL of N-GQD@V2O5 was mixed with various volumes of stock cysteine solution to get the final concentration of 0.1−125 μM. The resultant solutions were incubated for 2−14 min at room temperature under well-mixing conditions. After incubation at the specific time intervals, the change in fluorescence intensity was recorded at the wavelength of 460 nm under the excitation wavelength at 360 nm. The selectivity of the sensing system was evaluated by adding 22 different types of interfering solutions into N-GQD@V2O5 solution, and then the quenching of fluorescence was recorded after the incubation for 4 min. The measurements for the detection of all sensor solutions were performed in triplicate. Detection of Cysteine in Human Serum Samples. To evaluate the feasibility of using N-GQD@V2O5 sensor for biomedical application, the standard addition method was used to determine cysteine in human serum samples. Similar to the detection procedure used in PBS buffer solution, 2 mL of N-GQD@V2O5 solution was mixed with 100× diluted human serum solution and then various cysteine solutions were spiked into the mixture to obtain the concentration of 2−12 μM. The cysteine concentration in the serum sample was optically detected after 4 min of incubation. C


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Figure 2. (a) XRD patterns, (b) TEM image, and (c) dispersion of bulk and exfoliated V2O5 nanosheets in water, (d) TEM image, (e) HRTEM image, (f) lattice pattern of V2O5 (0.34 nm), and (g) N-GQDs (0.21 nm) of N-GQD@V2O5 nanocomposites. Cytotoxicity Assay. The cytotoxicity of N-GQD@V2O5 was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HeLa cell was incubated into 96-well plates at a concentration of 1 × 104 cells per well in 0.2 mL of Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and 1% penicillin. The cells were incubated at 37 °C overnight in an atmosphere of 5% CO2. The expended medium was then replaced by fresh medium containing 10−70 μg/mL of nanomaterials, including N-GQDs, as-prepared V2O5 nanosheets, and N-GQD@ V2O5 nanocomposites. The plates were further incubated at 37 °C for 6 h. After being washed with PBS twice, the 96-well plates were then filled with fresh DMEM (0.2 mL/well) and reincubated for additional 24 h. To determine the cell viability, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT reagent) was added into the 96well plate (100 mL per well) and then incubated again at 37 °C for 2 h. The absorbance of each well was determined by the microplate reader at 570 and 600 nm.

Figure 1c shows the emission spectra of N-GQDs under the different excitation wavelength of 310−410 nm. The emitted fluorescence intensity at 460 nm increases from 310 to 360 nm and then decreases when the excitation wavelength is in the range of 370−410 nm. These results clearly indicate that the emission wavelength of N-GQDs is completely independent of the excitation wavelength and the maximum emission occurs at 360 nm. The bright blue color of N-GQDs illustrated in the inset of Figure 1c clearly shows its fluorescence property under irradiation of a 360 nm UV lamp, which means that N-GQDs have the superior photoluminescence property for sensing of analytes because of the presence of carboxylic functional groups and nitrogen doping in GQDs surface.12,31 The XPS survey scan as well as the deconvoluted C 1s peak of N-GQDs is shown in Figure 1d. The survey scan clearly shows the characteristic peaks of C 1s (284.3 eV), N 1s (400.1 eV), and O 1s (531.7 eV) (inset of Figure 1d). The small nitrogen peak is contributed from the presence of N doping. The deconvoluted C 1s peak of as-prepared N-GQDs shows peaks of CC, C−N, and −COOH at 284.5, 287.8, and 288.5 eV, respectively, indicating the existence of graphitic carbon planes, carboxyl functional groups, and nitrogen doping. The nitrogen doping is further confirmed by the deconvoluted spectra of N 1s. As shown in Figure S1 (Supporting Information), two peaks centering at 398.8 and 401.6 eV are attributed to the pyridinic N and graphitic C−N functional groups, respectively, which proves that the doping is predominantly generated in the graphitic plane rather than amine linkage.32 The quantum yield of the fluorophore is a crucial parameter to develop a sensitive fluorometric sensing system. In this

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RESULTS AND DISCUSSION Characterization of the N-GQDs. To construct the NGQD@V2O5 sensing probe for cysteine detection, N-GQDs are first prepared through the hydrothermal method. The TEM image shows the spherical N-GQD nanoparticles with homogeneous distribution (Figure 1a). In addition, the fringes of the carbon lattice can be viewed clearly from the HRTEM image (inset of Figure 1a), which match well with the characteristic (100) plane of graphitic carbon.29,30 The particle size distribution of N-GQDs shown in Figure 1b is in the range of 2−8 nm, and the average lateral size is 4.7 ± 0.5 nm (n = 65). It is anticipated that such narrow-distributed particle size with superior homogeneous dispersion can significantly enhance the sensing properties. D


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Figure 3. (a) XPS survey spectra of as-synthesized V2O5 nanosheets, N-GQD@V2O5 nanocomposites, and deconvoluted XPS spectra of (b) V 2p, (c) C 1s, and (d) N 1s peaks of N-GQD@V2O5 nanocomposites.

confirms the successful nanodimensional property of ultrathin V2O5 nanosheets. In addition, the layer-to-layer distance (dspacing), calculated from (001) plane, is found to be 0.42 nm. The morphology of exfoliated V2O5 nanosheets was further examined using TEM images. As shown in Figure 2b, the exfoliated V2O5 exhibits two-dimensional layered nanosheet structures, clearly indicating that DMF is an effective delaminating agent for entering into the interlayer gallery for various layered materials.40 The smooth edges of nanosheets confirm the maintenance of integrity even after the prolonged sonication under high-frequency conditions. The image in Figure 2c shows the dispersion of bulk (i) and exfoliated (ii) V2O5 after addition to the distilled water for 2 min. This clearly indicates that bulk V2O5 precipitates rapidly within 2 min, whereas the V2O5 nanosheets disperse well in solution due to their nanostructures and induced hydrophilic functional groups. After deposition of N-GQDs onto V2O5 nanosheets, the spherical dots of N-GQDs onto the 2-D V2O5 nanosheets are clearly present in TEM and HRTEM images (Figure 2d,e). The lattice distances of 0.21 and 0.34 nm (Figure 2f,g), which correspond to N-GQDs29 and nano-V2O5,39 respectively, confirm the successful formation of N-GQD@V2O5 nanocomposites. It is noteworthy that the as-prepared V2O5 has little aggregation during the preparation procedures. As shown in Figure S2 (Supporting Information), the AFM image and the Z-axis height profiles exhibit the smooth sheet-like morphology, with a height of only about 0.8−1.7 nm, which corresponds to 2−4 layers of V2O5 nanosheets.

study, the quantum yield of N-GQDs, measured by using fluorescein as the standard,30,33 is optically determined to be 0.34, which is satisfactory to serve as the fluorometric sensing probe for biomolecule detection. Our previous study has fabricated the as-prepared GQDs by the pyrolysis of citric acid, and the quantum yield of 0.102 was reported.25 Several researches have reported that doping of GQDs with heteroatoms such as nitrogen (N) and sulfur (S) can improve the quantum yield of GQDs.31,34,35 Anh et al. used a one-pot hydrothermal method to synthesize N, S-GQDs for highly sensitive and selective detection of nanomolar Hg2+ and found that the doping of electron-rich N atoms can enhance the quantum yield of N, S-GQDs to 0.419.31 More recently, nitrogen-doped GQDs have been used for the detection of metal ions and cysteine. The reported quantum yield of NGQDs are in the range of 0.15−0.94,36−38 which is highly dependent on the precursor and method used for nitrogen doping. Characterization of V2O5 Nanosheets and N-GQD@ V2O5 Nanocomposites. The ultrathin V2O5 nanosheet, synthesized by exfoliation of bulk V2O5 with DMF, was further characterized by XRD and TEM. The XRD patterns of the exfoliated V2O5 nanosheets in Figure 2a show the strong diffraction peak at 20.2 and 26.0° 2θ, which can be assigned as the (001) plane of orthogonal V2O5. Another diffraction peak at 34.4° 2θ is the (002) plane of bulk V2O5 (JCPDS 411426).39 It is noteworthy that most of the crystalline planes of bulk V2O5 are diminished and the peak of (001) plane becomes weak and broad when V2O5 is exfoliated, which E


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Figure 4. (a) Influence of cysteine concentration on the fluorescence intensity of N-GQD, (b) the fluorescence emission spectra of N-GQD@V2O5 before and after the addition of cysteine (inset is the photograph of the corresponding color under 365 nm UV irradiation), (c) fluorescence spectra of N-GQDs in the presence of various concentrations of V2O5 nanosheets, and (d) the change in the fluorescence intensity ratio as a function of V2O5 concentration.

Cysteine Detection Mechanism by N-GQD@V2O5. The applicability of the newly developed fluorescence turn off−on N-GQD@V2O5 nanocomposites is evaluated by the addition of cysteine. As shown in Figure 4a, the addition of 0−200 μM cysteine in N-GQDs has ignorable influence on the change in fluorescence intensity, confirming the absence of electronic interactions between N-GQDs and cysteine. Interestingly, upon deposition of N-GQDs to V2O5 nanosheets, the fluorescence intensity of N-GQDs is distinctly quenched within 10 min (Figure 4b), which is mainly attributed to the electrostatic interaction between electrophilic V2O5 nanosheets and nucleophilic N-GQDs. However, the addition of cysteine into N-GQD@V2O5 nanocomposites regenerates the fluorescence intensity up to 80% of its initial value (inset of Figure 4b). The recovery of fluorescence intensity is based on the redox reaction between cysteine and V2O5. As depicted in Scheme 1, the added cysteine can serve as the reducing agent to convert V5+ in V2O5 into V4+, whereas cysteine will simultaneously be oxidized to its dimeric cystine. The reduction of V2O5 releases the adsorbed N-GQDs from the N-GQD@V2O5 and subsequently results in the recovery of fluorescence intensity. To further confirm the effectiveness of using N-GQDs as the sensing probe, the cysteine detection by as-prepared GQD@ V2O5 was conducted. As illustrated in Figure S4 (Supporting

Figure 3a shows the XPS of full survey spectra of as-prepared V2O5 nanosheets and N-GQDs@V2O5 nanocomposites. It clearly shows the characteristic peaks of C 1s (284.2 eV), V 2p (516.9 and 530.2 eV), and O 1s (531.2 eV). In addition, no N 1s peak is observed in the survey scan of V2O5. After the addition of N-GQDs, the N 1s peak at around 400 eV appears and the peak intensity of C 1s also increases, indicating the successful attachment of N-GQDs onto V2O5 nanosheets. The peak deconvolution of V 2p shows the binding energy of 2p3/2 and 2p1/2 at 517.1 and 523.9 eV, respectively, which is the characteristic peak of V5+ in V2O5.41 The deconvoluted C 1s spectra of N-GQD@V2O5 show a broad peak at 284.2 eV, which is attributed to CC functional groups of N-GQDs. Two other small peaks at 286.8 and 288.1 eV can be assigned as C−N and C−O, confirming the presence of doped nitrogen and carboxyl functional groups of N-GQDs in the nanocomposites.42 In addition, the deconvolution of N 1s spectrum (Figure 3d) indicates the presence of pyridinic (399.1 eV) and graphitic (401.6 eV) nitrogen,29 which is in good agreement with the as-synthesized N-GQD spectra shown in Figure S2. Moreover, the deconvoluted peak of O 1s at 528.9 eV is the V−O functional group of V2O5, whereas 531.1 eV is ascribed to the abundant hydroxyl groups (O−H) of N-GQDs (Figure S3, Supporting Information), further confirming the fabrication of N-GQD@V2O5 nanosheets. F


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Figure 5. (a) Fluorescence spectra of N-GQD@V2O5 in the presence of various concentrations of cysteine ranging from 0 to 125 μM, (b) the relationship between change in the fluorescence intensity ratio as a function of cysteine concentration from 0 to 125 μM (inset is the selected linear relationship in the low concentration range of 0−15 μM) and (c) fluorescence intensities of the fluorescent turn on N-GQD@V2O5 sensors in the presence of 22 different interfering substances.

Information), the fluorescence quenching of as-prepared GQDs upon addition of V2O5 is less than that of N-GQD@ V2O5 nanosheets, depicting the high affinity of N-GQDs onto V2O5 nanosheet surface. After addition of cysteine to the GQDs@V2O5 nanocomposites, the recovered fluorescence intensity is also less in comparison to that of the N-GQD@ V2O5. This means that N-GQD@V2O5 can effectively serve as a promising sensing platform for cysteine detection, where NGQDs function as the fluorometric probe and V2O5 nanosheets act bifunctionally as the fluorescence quencher and cysteine recognizer.43,44 To ascertain the reaction mechanism and quenching effect on the fluorescence intensity of N-GQDs in the presence of V2O5, various concentrations of dispersed V2O5 nanosheets were added to the identical aliquot of N-GQDs. Figure 4c shows the fluorescence intensity of N-GQDs in the presence of various concentrations of V2O5 ranging from 5 to 60 μg/mL. The fluorescence intensity decreases gradually with the increase in V2O5 concentration. The decrease in fluorescence intensity is attributed to the adsorption of N-GQDs on the surface of V 2O5 nanosheets, which restricts the π−π conjugation of N-GQDs because of the electrostatic interaction of the metal oxide framework of V2O5.45

Furthermore, the change in fluorescence intensity as a function of V2O5 concentration ranging from 0 to 60 μg/mL exhibits a linear relationship with a correlation coefficient of 0.976 (Figure 4d). On the basis of this finding, 60 μg/mL is selected as the optimal V2O5 concentration for the subsequent experiments. Optimization and Detection of Cysteine. The effect of pH and reaction time on cysteine detection by N-GQD@V2O5 was further optimized. Figure S5a (Supporting Information) shows the effect of pH on the fluorescence intensity of NGQD@V2O5 probe in the presence of 50 μM cysteine. The change in fluorescence intensity ratio (FR/FR0) of N-GQD@ V2O5 upon addition of cysteine enhances with the increase in pH from pH 5.0 to 6.5, which is mainly due to the fact that the sulfhydryl group in cysteine can be easily deprotonated to lower the nucleophilicity in the pH range examined. Alternatively, the stability of V2O5 framework increases as the pH increase to > 7, which would reduce the redox reaction rate of V2O5 with cysteine. Therefore, a gradual decrease in the intensity ratio is also noted when the pH increases to 7.0−8.0. As illustrated in Figure S5b (Supporting Information), the fluorescence intensity increases rapidly with the increase in reaction time upon addition of 50 μM cysteine into the NG


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ACS Applied Materials & Interfaces GQD@V2O5 solution. The fluorescence intensity reaches the plateau after 4 min of incubation, indicating the fast-response characteristics of N-GQD@V2O5 nanocomposites toward cysteine detection. Figure 5a illustrates the efficiency of turn off−on fluorescence of N-GQD@V2O5 sensor on cysteine detection. Under optimal conditions, fluorescence intensity of N-GQDs is originally turned off with the addition of 60 μg/mL of V2O5 nanosheets and subsequently turned on by the addition of cysteine in the concentration range of 0−125 μM after 4 min of incubation. Figure 5b shows the change in the fluorescence intensity ratio as a function of cysteine concentrations. A twostage linear relationship between the change in fluorescence intensity and cysteine concentration is observed. The fluorescence intensity increases rapidly as the cysteine concentration increases from 0.1 to 15 μM linearly and then makes a slight increase up to 125 μM. Because the reaction of cysteine with N-GQDs@V2O5 nanocomposites is a surfacemediated reaction and cysteine molecules need to diffuse to the V2O5 surface first, the rate of redox reaction between V2O5 and cysteine is rapid in the low concentration range because of the abundant availability of N-GQD@V2O5 nanosheets. After a certain time of reaction, however, the availability of V2O5 nanosheet decreases and the slope of recovered fluorescence becomes flat at a high cysteine concentration of 15−125 μM, which resembles the typical heterogeneous reaction.46,47 A good linear relationship over the range of 0.1−15 μM with the correlation coefficient (r2) of 0.994 is also clearly obtained (inset in Figure 5b). In addition, the limit of detection (LOD), determined by the 3σ/S (σ is the standard deviation of the lowest signal and S is the slope of linear calibration plot) is 50 nM. Efficient selectivity is a very important parameter for the successful application of the sensing system. Hence, the selectivity of N-GQD@V2O5 sensor was analyzed in the presence of interfering molecules and the results are presented in Figure 5c. Because cysteine is a mostly common amino acid in human serum, a wide variety of interferences presented in blood, including metal ions (Mg2+, Mn2+, Cu2+, Fe3+, and Zn2+), inorganic salts (Na+, K+, and Ca2+), amino acids (aspartic acid, tyrosine, glutathione, and methionine), sugar (glucose and fructose), reducing agents (citric acid, ascorbic acid, glutamic acid, and uric acid), and proteins (bovine serum albumin, tyrosinase, acetyl cholinesterase (AChE), and glucose oxidase (GOx)) were tested. The concentration is 100 μM for protein and 500 μM for other interferences, which is 2−10 times higher than that of spiked cysteine (50 μM). It is clear that the recovery of fluorescence intensity of N-GQD@V2O5 system in the presence of most interfering species is