N-Doped Graphene Quantum Dots-Decorated V2O5 Nanosheet for

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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 ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b15120 • Publication Date (Web): 11 Dec 2017 Downloaded from http://pubs.acs.org on December 13, 2017

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ACS Applied Materials & Interfaces

N-doped Graphene Quantum Dots Decorated V2O5 Nanosheet for Fluorescence Turn Off-On Detection of Cysteine Akhilesh Babu Ganganboina1, Ankan Dutta Chowdhury2, Ruey-an Doong1, 2,*

1 Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, 101 Section 2, Kuang-Fu Road, Hsinchu, 30013, Taiwan 2 Institute of Environmental Engineering, National Chiao Tung University, 1001 University Road, Hsinchu, 30010, Taiwan.

ABSTRACT The development of fast-response sensing technique for detection of cysteine can provide an

analytical platform for pre-screening of disease. Herein, we have developed a fluorescence turn offon fluorescence sensing platform by combining nitrogen-doped graphene quantum dots (N-GQD) 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 ACS Paragon Plus Environment

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nanosheets. Addition of cysteine triggers the reduction of V2O5 to V4+ as well as the release of NGQDs 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 µM 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.

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, AIDS, 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 ACS Paragon Plus Environment

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However, these techniques need expensive instruments, skilled persons and tedious procedures for sample preparation, which limit their application. Hence, there is 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 sensor 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 multi-walled carbon nanotubes hybrid for electrochemical detection of cysteine.7 Although these methods can reach 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 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 N-doped graphene quantum dots (N-GQDs) have emerged as a new class of photo-stable 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 application such as fluorescence sensors,12-17 bio-imaging18-20 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 analytes detection.22 Twodimensional MnO2 and MoS2 nanosheets can react with GQDs through surface functional groups or surface adsorption to quench the fluorescence intensity of GQDs.23,

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The layered vanadium

pentoxide (V2O5) nanomaterial has the excellent properties such as redox-activity, wide optical band gap, 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 ACS Paragon Plus Environment

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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 presence of cysteine.27 It is interesting to note that the N-GQDs have adsorption affinity toward V2O5 surface. The attachment of N-GQDs 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 nanosheets materials in the presence of cysteine. However, the combination of N-GQDs with V2O5 as the turn off-on fluorescent sensor have received less attention and the application of N-GQD@V2O5 fluorescent sensor for determination of cysteine has not been reported.

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Scheme 1. Schematic illustration of N-GQD@V2O5 nanocomposites preparation and mechanism for cysteine detection. Herein, we have developed a fluorescence turn off-on N-GQD@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 ACS Paragon Plus Environment

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when N-GQDs are adsorbed onto ultrathin V2O5 nanosheets. The role of V2O5 nanosheets in the sensing platform is to serve as both fluorescence nano-quencher 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 limit of detection (LOD) of 50 nM within 4 min. In addition, the NGQD@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 cysteine-associated disease monitoring and diagnosis in biological applications.

EXPERIMENTAL 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 bi-distilled 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. 100 mg of bulk V2O5 powder were added to 200 mL of DMF solutions and the mixture was shaken over night at room temperature to suspend the V2O5 powder. The resultant suspension was then sonicated at room temperature for 3 d. After ultrasonic treatment, 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 ACS Paragon Plus Environment

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from DMF. The obtained V2O5 nanosheet was washed several times with ethanol to remove residual DMF, and then dried in 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 citric acid and 0.18 g urea were added to 5 mL of bi-distilled deionized water and stirred to form a clear solution. The solution was then transferred into a 20-mL of Teflon lined 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 5,000 rpm for 5 min. The solid was then dispersed in water and dialyzed in a 1 kDa dialysis bag for 24 h, which 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 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 as prepared V2O5 nanosheets, N-GQDs and N-GQD@V2O5 were characterized using JEOL JEM-ARM200F transmission electron microscope (TEM), JEOL JEM-2010 high-resolution transmission electron microscopy (HRTEM) at 300 kV and Bruker Dimension Edge atomic force microscope (AFM) were used. X-ray diffraction (XRD) patterns were recorded using Bruker D8 Xray 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-GQDs based composites were recorded by Hitachi F-7000 fluorescence spectrophotometer. Detection of Cysteine. The detection of cysteine using N-GQD@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 were 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 ACS Paragon Plus Environment

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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 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 NGQD@V2O5 sensor for biomedical application, 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 were 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 serum sample was optically detected after 4 min of incubation. Cytotoxicity assay. The cytotoxicity of N-GQD@V2O5 was examined by MTT assay. HeLa cell was incubated into 96-well plates at a concentration of 1 × 104 cells per well in 0.2 mL of DMEM medium containing 10 % FBS 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 NGQD@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 96-well 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.

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Characterization of the N-GQDs. To construct the N-GQD@V2O5 sensing probe for cysteine detection, N-GQDs are first prepared through the hydrothermal method. The TEM image shows the spherical N-GQDs nanoparticles with homogeneous distribution (Figure 1a). In addition, the fringes of carbon lattice can be viewed clearly from 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.

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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.

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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 on 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 360nm 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 prove 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 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 sulphur (S) can improve the quantum yield of GQDs.31, 34-35 Anh et al. used one-pot hydrothermal method to synthesize N, SACS Paragon Plus Environment

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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 0.419.27 More recently, Ndoped GQDs have been used for the detection of metal ions and cysteine. The reported quantum yield of N-GQDs 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 shows 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 41-1426).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 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.

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Figure 2. The (a) XRD patterns, (b) TEM image and (c) dispersion of bulk and exfoliated V2O5 nanosheets in water, (d) TEM image, (e) HRTEM image and (f) lattice pattern of V2O5 (0.34 nm) and (g) N-GQDs (0.21 nm) of N-GQD@V2O5 nanocomposites. The morphology of exfoliated V2O5 nanosheets was further examined using TEM images. As shown in Figure 2b, the exfoliated V2O5 exhibits 2-dimensional layered nanosheet structures, clearly indicating that DMF is as an effective de-laminating agent to enter 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 nano-structures 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 (Figures 2d and 2e). The lattice distances of 0.21 nm and 0.34 nm (Figures 2f and 2g), which correspond to N-GQDs29 and nanoV2O5,39 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 height of only about 0.8 – 1.7 nm, which corresponds to the 2 – 4 layers of V2O5 nanosheets. Figure 3a shows the XPS of full survey spectra of as-prepared V2O5 nanosheets and NGQDs@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 eV 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 ACS Paragon Plus Environment

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groups of N-GQDs. Another two 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-GQDs spectra shown in Figure S2. Moreover, the deconvoluted peak of O 1s at 528.9 eV is the V-O functional group of V2O5, while 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.

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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 NGQD@V2O5 nanocomposites. 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 ACS Paragon Plus Environment

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shown in Figure 4a, 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+, while cysteine will simultaneously oxidized to its dimeric cystine. The reduction of V2O5 releases the adsorbed N-GQDs from the NGQD@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 Information), the fluorescence quenching of as-prepared GQDs upon addition of V2O5 is less than that of N-GQDs@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 with the N-GQD@V2O5. This mean that the N-GQD@V2O5 can effectively serve as a promising sensing platform for cysteine detection where N-GQDs function as the fluorometric probe and V2O5 nanosheets act bi-functionally as the fluorescence quencher and cysteine recognizer.43, 44 In order 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 V2O5 nanosheets, which ACS Paragon Plus Environment

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restricts the π-π conjugation of N-GQDs because of the electrostatic interaction of 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). Based on this finding, 60 µg/mL is selected as the optimal V2O5 concentration for the subsequent experiments. (a)

FL intensity (a.u.)

1250

1500

FL intensity (a.u.)

FL intensity (a.u.)

1750

1000 750 500

0 µM Cysteine 1250 1000

200 µM Cysteine 750 500 250 0

250 0

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after cysteine addition

600 400

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60 µg/mLV2O5 1000 800 600 400

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Change in fluorescence (F/F0)

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Wavelength (nm)

(d)

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.7 2

R =0.976

0.6 0.5 0.4 0.3 0.2

Y=0.02x+0.035

0.1 0.0

0

10

550

Wavelength (nm)

20

30

40

50

60

V2O5 (µg/mL)

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 fluorescence intensity ratio as a function of V2O5 concentration. 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 ACS Paragon Plus Environment

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effect of pH on the fluorescence intensity of N-GQD@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 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 N-GQD@V2O5 solution. The fluorescence intensity reaches the plateau after 4 min of incubation, indicating the fast response characteristics of NGQD@V2O5 nanocomposites toward cysteine detection. Figure 5a illustrates the efficiency of turn off-on fluorescence of N-GQD@V2O5 sensor on cysteine detection. Under the 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 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. Since the reaction of cysteine with N-GQDs@V2O5 nanocomposites is a surface-mediated 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 become flat at high cysteine concentration of 15 – 125 µM, which is resemble to the typical heterogeneous reaction.46, 47 A good linear relationship over the range of 0.1 to 15 µM with the correlation coefficient (r2) of 0.994 is also clearly obtained (Inset in Figure 5b). In


15


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.

1600

(b) 1.8

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125 µM

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at hi C one ys te in e

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% Change in fluorescence (FR/FRo)

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FL intensity (a.u.)

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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 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 to 15 µM) and (c) fluorescence intensities of the fluorescent turn on N-GQD@V2O5 sensors in the presence of 22 different interfering substances. Efficient selectivity is a very important parameter for the successful application of 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. Since cysteine is a mostly common amino acid


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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 (BSA), 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 lower than < 10%. Although glutathione and aspartic acid have relatively high fluorescence intensity (16 – 23%) in comparison with other interference species, the response is still much lower than that of cysteine. In spite of similar functional behaviour between cysteine and glutathione, the size of cysteine molecule is smaller than glutathione. This would allow the small thiol group on cysteine to interact with V2O5 nanosheets surface more rapidly, and results in the enhancement of fluorescence recovery more readily. In addition, the fluorescence intensity shows obvious recovery only after the addition of cysteine, confirming the excellent selectivity of N-GQD@V2O5 towards cysteine. Hence, this nanocomposite is capable of overcoming interference from common electrolytes and biological species for efficient and selective detection of cysteine. Several studies have developed the turn off-on sensors for detection of cysteine by carbonbased quantum dots in the presence of metal ions such as Hg2+, Cu2+ and Fe3+ ions.36, 38, 48-51 Zheng et al. have used carbon nitride nanosheets to detect cysteine in the presence of Hg2+ and then the developed turn off-on sensor was applied to tap water sample for Hg2+ and cysteine analyses.49 Ndoped carbon dots were also developed for cysteine detection in the presence of Hg2+ and low LOD values of 0.79 – 45.8 nM were obtained for cysteine.50, 52 In addition, a turn off-on fluorescence method based on N-GQDs and Hg2+ was reported for the detection of biothiols including cysteine and glutathione and found that the linear range of both biothiols is in the range of 0.5 – 5 µM with LODs of 36 and 34 nM for cysteine and glutathione, respectively.38 It is noteworthy that the detection mechanism for these works are mainly based on the well-known Hg2+-cysteine interaction ACS Paragon Plus Environment

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where the fluorescence intensity of carbon-based quantum dots can be quenched by toxic Hg2+ and subsequent regeneration by cysteine addition. However, the selectivity of the reported turn-off sensors based on Hg2+-cysteine principle is not high for other biothiols such as glutathione and homocysteine due to the strong affinity and rapid conjugation of Hg2+ with thiol groups. In addition, these sensors are hardly used in biological system because of the toxicity of Hg2+. More recently, Wu et al. have developed the N, S-codoped GQDs for the detection of cysteine in the presence of Fe3+ and found that the N, S-codoped GQD based sensors has good selectively toward cysteine detection with the LOD value of 540 nM.51 In this study, we have successfully developed the metal ion-free N-GQD@V2O5 nanosheet probes for the fluorescence turn off-on detection of cysteine based on the oxidation-reduction interaction between V2O5 nanosheets and cysteine. The NGQD@V2O5 gives an excellent linear and wide calibration range of 0.1 – 125 µM with LOD of 50 nM and superior selectivity of cysteine over other biothiols. Table S1 (Supporting Information) summarizes the analytical performance of cysteine by different fluorescent sensing probes. It is clear that most reported sensing probes have a dynamic range of 1 – 2 orders of magnitude with LOD value of 20 – 1200 µM.51-60 In this study, the N-GQD@V2O5 nanosheets are highly sensitive toward cysteine detection and the dynamic range can be up to 3 orders of magnitude with LOD of 50 nM, which is better than most of the reported data shown in Table S1. Furthermore, the nontoxic usage of V2O5 nanosheets and N-GQDs without any further functionalization make this new class of fluorescence turn off-on probe applicable for the selective and sensitive detection of cysteine in biological samples.

MTT assay and cysteine detection in human serum samples by N-GQD@V2O5. The biocompatibility of N-GQD@V2O5 nanosensor was evaluated using MTT assay and the applicability of the N-GQD@V2O5 nanosensor to biomedical application was examined by human serum samples. HeLa cells were incubated with various concentrations of as–prepared V2O5 nanosheets, NGQDs, and N-GQDs@V2O5 nanosensor for 24 h. Figure 6 shows cell viability of HeLa cells after incubating with V2O5 nanosheets, N-GQDs, and N-GQDs@V2O5 nanocomposites. The HeLa cell viability can maintain higher than 90% at 10 – 70 µg/mL N-GQDs, showing that N-GQDs have little ACS Paragon Plus Environment

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toxic effect on HeLa cells. Addition of V2O5 nanosheet-based has a slight effect on HeLa cell and higher than 75% of cell variability is observed when the added concentrations of V2O5-based nanomaterials are in the range of 10 – 70 µg/mL. These results indicate that the N-GQDs@V2O5 nanocomposite is biocompatible and can be used in biomedical application for the sensitive and selective analysis of cysteine in human serum samples.

N-GQDs

Nano-V2O5

0 10 30 50 70

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Cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


N-GQDs@V2O5

80

60

40

20

0

0 10 30 50 70

Concentration (µg/mL) Figure 6. Cell viability of HeLa cells after treatment of N-GQDs, V2O5 nanosheets, and NGQD@V2O5 nanocomposites at concentrations of 0 – 70 µg/mL. HeLa cells were incubated with nanomaterials for 1 h first, washed with PBS buffer, placed into fresh cell medium, and then reincubated for additional 24 h before MTT assay The analytical performance of fluorescence turn off-on N-GQD@V2O5 nanosensors was further evaluated by standard addition method in human serum samples. Various concentrations of cysteine ranging from 2 – 12 µM were spiked into the diluted human serum. Table 1 shows the analytical performance of fluorescence turn off-on N-GQD@V2O5 nanosensors for the detection of cysteine in human serum samples. The recoveries of the spiked cysteine concentration are in the


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range of 95 – 108% with a relative standard deviation (RSD, n = 5) of 1.7 – 7.0%, clearly indicating a good analytical performance for cysteine in biological matrix. Table 1. The analytical performance of fluorescence turn off-on N-GQD@V2O5 nanosensors for the detection of cysteine in diluted human serum samples.

Sample

Human serum samples

Spiked (µM)

Found (µM)

Recovery (%)

RSD (%) n=5

2

1.9

95

3.8

4

4.1

103

1.7

8

8.1

101

4.3

10

10.5

105

7.0

12

12.9

108

2.4

CONCLUSIONS In this study, we have prepared exfoliated V2O5 nanosheets through a simple and scalable liquid

method in the presence of DMF. The 2 – 8 nm of highly fluorescence N-GQDs at quantum yield of 0.34 were then deposited onto V2O5 nanosheets to serve as fluorescence turn off-on sensor for highly selective and sensitive detection of cysteine. Unlike the toxic metal ions quencher using Hg-thiol complexation principle to detect cysteine, the V2O5 nanosheets play a dual-function of nanoquencher and recognizer, while N-GQDs with quantum yield of 0.34 serve as the fluorescent probe for cysteine detection. Based on the redox reaction between V2O5 and cysteine, the developed turn off-on N-GQDs@V2O5 nanoprobes can be effectively detected cysteine in the concentration range of 0.1 – 125 µM with 2-stage linear relationship and the LOD of cysteine can be lower to 50 nM. In addition, the N-GQD@V2O5 can be applied for analysis of cysteine in spiked serum samples and the recovery is the range of 95 ± 3.8% to 108 ± 2.4% at 2 – 12 µM cysteine. Since no toxic metal ions nor further functionalization of V2O5 and N-GQDs is required, the fluorescence turn off-on NACS Paragon Plus Environment

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GQD@V2O5 platform has clearly demonstrated its superiority on specific detection of cysteine in complex matrix, which can pave an avenue for development of cost effective, environmentally friendly and robust sensing probes for detection and diagnosis of cysteine in biomedical applications.

ASSOCIATED CONTENT

Supporting Information Deconvoluted N 1s spectrum of N-GQDs; AFM image of as-prepared V2O5; O 1s XPS spectrum of N-GQDs@V2O5; fluorescence emission spectra of GQD@V2O5 and effect of pH and incubation time on cysteine detection using N-GQDs@V2O5. This information is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author Corresponding

author:

Ruey-an

Doong

(Email:

[email protected];

[email protected], Tel: +886- 3-5726785, Fax: +886-3-5725958.)

Notes The authors declare no competing financial interests.

ACKNOWLEDGEMENTS The authors thank the Ministry of Science and Technology (MOST), Taiwan for financial

support under grant Nos. MOST 104-2221-E-009-020-MY3 and 105-2113-M-009-023-MY3.

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1400

(i) (ii) (iii)

(iii)

1200

1.8

1.5

1000

(ii)

800 600

1.3

400 200 0

0

(i)

1600

FL Intensity (a.u)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46



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1.0

400

450

500

550

600

0

Wavelength (nm)

20

360 nm

460 nm

DMF N-GQDs

Exfoliation Bulk V2O5

2

V2O5 Nanosheet Oxidation

+

+ 2H + 2e

+ 2 H++2 eACS Paragon Plus Environment

40

60

80

100

Cysteine concentration (M)

-

120

N-Doped Graphene Quantum Dots-Decorated V2O5 Nanosheet for

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