Nitrogen and Sulfur Codoped Reduced Graphene Oxide as a General

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Nitrogen and Sulfur Co-doped Reduced Graphene Oxide as a General Platform for Rapid and Sensitive Fluorescent Detection of Biological Species Lu Chen, Liping Song, Yichi Zhang, Ping Wang, Zhidong Xiao, Yu-Guo Guo, and Feifei Cao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01030 • Publication Date (Web): 18 Apr 2016 Downloaded from http://pubs.acs.org on April 19, 2016

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ACS Applied Materials & Interfaces

Nitrogen and Sulfur Co-doped Reduced Graphene Oxide as a General Platform for Rapid and Sensitive Fluorescent Detection of Biological Species Lu Chen1,ǂ, Liping Song1,ǂ, Yichi Zhang1,2, Ping Wang1, Zhidong Xiao1,*, Yuguo Guo2 and Feifei Cao1,* 1

College of Science, Huazhong Agricultural University, Wuhan, 430070, People’s Republic of China 2

Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, People’s Republic of China E-mail: [email protected] [email protected] ǂ These authors contributed equally to this work. ABSTRACT: Nitrogen (N) and sulfur (S) co-doped reduced graphene oxide (N,S-rGO) was

synthesized through a facile solvothermal process. The introduction of N and S heteroatoms into GO effectively activated the sp2-hybridized carbon lattice and made the material an ideal electron/energy acceptor. Such unique properties enable this material to perform as a general platform for rapid and sensitive detection of various biological species through simple fluorescence quenching and recovering. When quantum dot (QD)-labeled HBV (human being diseases related genes hepatitis B virus DNA) and HIV (human being diseases related genes human immunodeficiency virus DNA) molecular beacon probes were mixed with N,S-rGO, QDs fluorescence was quenched; when target HBV and HIV DNA were added, QDs fluorescence was recovered. By the recovered fluorescence intensity, the target virus DNA detection limits were reduced to 2.4 nM for HBV and 3.0 nM for HIV with detection time of less than 5 min. It must be stressed out that different viruses in the same homogenous aqueous media could be discriminated and quantified simultaneously through choosing diverse QDs probes with different colors. Moreover, even one mismatched target DNA could be distinguished using this method. When altering the molecular beacon loop domain to protein aptamers, this sensing strategy was also able to detect thrombin and IgE in 5 min with detection limits of 0.17 ng mL-1 and 0.19 ng mL-1 respectively, which was far more rapid and sensitive than bare GO-based fluorescence detection strategy.

KEYWORDS: Nitrogen and Sulfur Co-doping; Reduced Graphene Oxide; Quantum Dot; Biomolecules Sensing; Virus DNA Detection

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1. INTRODUCTION Graphene oxide (GO), a newly developed two-dimensional carbon material, has attracted intensive attentions due to its relatively good electronic, thermal and mechanical properties.1-3 It is employed as a catalyst in many electrochemical biosensors.4-5 Meanwhile, it is also used as a good luminescence quencher to construct many optical sensing systems.6-7 Yang et al. have reported a DNA detection method by exploring water soluble GO as a fluorescent quencher for the first time in 2009.8 After that, there are a lot of reports about the GO application in fluorescence and chemluminescnece sensing.9-11 For instance, Gao et al. have reported a polyethylene glycol protected GO-based protein detection method by aptamers, and decreased the detection limit to 0.0048 nM.12 Kanaras’s group has demonstrated a DNA sensor by up-conversion nanoparticles and GO in which excellent energy and electronic transfer is proved between nanoparticles and GO.13 Ju et al. have employed GO as a fluorescence quencher to realize multiplex DNA detection and demonstrated it was a novel platform for effective sensing of biomolecules by fluorescence resonance energy transfer from quantum dots (QDs) to GO.14,15 Our previous work has demonstrated that GO can be employed as a good quencher towards QDs to fabricate fluorescent sensor for protein and virus detection.16 However, among those GO-based DNA and protein detection methods, there are still some drawbacks such as relatively long detection time, tedious operation steps and limited detection sensitivity leading to limited applications. This may be mainly caused by two factors: first, the electron transfer efficiency between electron donors to GO is decreased owing to the inertness of carbon atoms; second, biomolecules may absorb onto the surface of GO by non-specific interactions leading to a negative influence towards the specific interaction between the probe and the target.17-19 To solve this problem, heteroatom doping is an excellent strategy to break the inertness of the GO layer and to modulate the electronic and chemical properties by regulating the electronic states within the GO sheet.20 Many efforts have been devoted to co-doping multiplex heteroatoms because it may synergistically create unique electronic structure on the sp2 hybridized carbon. In searching of promising candidates for heteroatom co-doping, nitrogen (N) and sulfur (S) have been proved to be effective elements to significantly improve the activity of bare GO.21-24 It has been proved that N, S co-doped GO with an optimal sulfur loading can effectively break the inertness of carbon material, activate the sp2-hybridized carbon lattice as well as facilitate the electron transfer from


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electron donor to GO sheets.25 So intensive efforts have devoted to search the simple way for N, S co-doped graphene or GO preparation. Recently, Huang et al. have prepared N, S co-doped graphene for electrochemical application by a one-step co-doping method.26 It is still highly desired to explore a more facile N, S co-doping GO route. Based on these motivations, we synthesized N, S co-doped reduced graphene oxide (N,S-rGO) by a simple and effective solvothermal method for sensing DNA and proteins. Previous studies have proved that GO can quench QDs fluorescence with a relatively high efficiency. However, the binding between QDs and N,S-rGO would become stronger due to the synergistic effect brought by N, S co-doping, since nitrogen and sulfur can endow the GO surface with more negative charge. To demonstrate the issue, molecular beacon structured HBV and HIV probes were designed by modifying 5’ end with green-color CdSe QDs and red-color CdSe QDs via streptavidin-biotin interaction. N,S-rGO was adopted as a fluorescence quencher toward the virus probe. When target viruses were introduced into the probe solution, the target induced fluorescence recovery took place by DNA hybridization. By the recovery fluorescence intensity, the HBV and HIV were discriminated and quantified (Scheme 1). Moreover, to prove the effectiveness of this sensing strategy in protein detection, the molecular beacon loop domain was changed to thrombin and IgE aptamers. By the aptamer-target binding induced fluorescence recovery, thrombin and IgE can be detected with a detection limit of 0.17 ng mL-1 and 0.19 ng mL-1, respectively, which is significantly lower than that of bare GO based fluorescent method. The results have demonstrated that N,S-rGO can provide a better optical platform than bare GO for biomolecules sensing.

2. EXPERIMENTAL SECTION 2.1 Materials and Reagents All chemicals were of analytical grade and used without any further purification. Sodium phosphate monobasic dihydrate (NaH2PO4·2H2O), sodium phosphate dibasic dodecahydrate (Na2HPO4·12H2O), sodium chloride (NaCl) and thrombin as well as IgE were commercially available from Sigma Aldrich (St. Louis, MO), 2-aminothiophenol (C6H7NS) was purchased from Aladdin Reagent Company. 525 nm and 623 nm streptavidin modified quantum dots (SA-QDs) were purchased from Wuhan Jiayuan Quantum Dots Company (Wuhan). All other reagents were



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purchased from Sinopharm Chemical Reagent Co., Ltd. (China). 2.2 Synthesis of GO and N,S-rGO GO was prepared from graphite powder by a modified Hummers’ method. The N,S-rGO was prepared through a facile modified one-pot solvothermal method.24 In brief, 400 mg as-prepared GO powder was dissolved into 200 mL deionized water to form 2.0 mg mL-1 GO aqueous solution. The solution was ultrasonicated for 1 h and centrifuged at 1000 rpm for 15 min to remove the unexfoliated particles. Afterwards, 1.5 mL of 2-aminothiophenol was added into the GO aqueous solution and ultrasonicated for another 5 min. Polyphosphoric acid (H6P4O13) was then added into the above solution under sustainable stirring. The solution was refluxed at 80 °C for 24 h and cooled to room temperature. The obtained mixture was filtered and washed with ethanol and deionized water for five times. The black product was dried at 80 °C for 24 h and next annealed at 650 °C for 30 min with a heating rate of 5 °C min-1 under argon flow. Finally, the N,S-rGO was obtained after cooling down to room temperature naturally. 2.3 Fluorescence measurement for biomolecules detection In a typical HBV and HIV fluorescence detection procedure, firstly, 1.010-6 M 525 nm SA-QDs and 1.010-6 M HBV biotin modified capture DNA were mixed well to form QDs-P1. 1.010-6 M 623 nm SA-QDs and 1.010-6 M HIV biotin modified capture DNA were mixed well to form QDs-P2. After that, 2 µL QDs-P1 and QDs-P2 as well as 60 ng mL-1 N,S-rGO were added into the 0.01 mol L-1 PBS buffer solution (pH 8.0). Finally, different concentrations of HBV and HIV DNA were added into the solution to form a 600 µL detection sample and then recorded the fluorescence spectra. The excitation wavelength was 388 nm and the maximum emission wavelength was 525 nm and 623 nm. The excitation and emission slits were both 10 nm. The thrombin and IgE detection procedure was under the same way. The serum samples detection was performed under the same steps with only one difference that 10 µL serum was added into the buffer solution before volume was set at 600 µL by 0.01 mol L-1 PBS solution at ambient conditions. 2.4 Kinetic determination of fluorescence quenching reaction The kinetic detection procedure of HBV DNA was below. Firstly, 2 µL QD-P1 was added into the 0.01 mol L-1 PBS buffer solution (pH 8.0) to form a 600 µL detection sample containing 360 µL 0.1 µg mL-1 N,S-rGO. For another equivalent detection sample, 2 µL QD-P1 was mixed


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with 2 µL HBV DNA for 30 min at 37℃ before adding 360 µL 0.1 µg mL-1 N,S-rGO. The quenching effect of the two samples was detected in 140 min. The kinetic determination was repeated in the 1% human serum samples at the same condition. The kinetic detection procedure of HIV DNA was under the same way. 2.5 Characterization. Transmission electron microscopy (TEM) and High-resolution TEM (HRTEM) images were observed on JEM-21001F (JEOL). Scanning electron microscopy (SEM) image was recorded on JSM-6390LV (Japan). X-ray diffraction (XRD) measurements were carried out using a Rigaku D/max-2500 diffractometer with filtered Cu Kα radiation (λ=1.54056 Å). The X-ray photoelectron spectroscopy (XPS) analysis was performed on the ESCALab 250Xi (Thermo Scientific). Raman measurements were recorded on a DXR from Thermo Scientific using laser excitation at 532 nm. FTIR spectra were collected by a NEXUS 670 FTIR spectrometer using KBr disks. The PL spectra were recorded by the RF-5301 PC (Shimdzu). 3. RESULTS AND DISCUSSION 3.1 Morphology and Structure Characterization GO prepared by a modified Hummers’ method exhibited sheet structure with partly folds, which was similar to the previous report (Figure S1).14 The as-prepared N,S-rGO can still maintain initial ultrathin layered morphology of GO even through nitrogen and sulfur co-doping procedure (Figure 1A). HRTEM image of N,S-rGO (inset in Figure 1A) shows clear crystalline planes of carbon (002) with a distance spacing of 0.35 nm. The larger lattice fringe could be ascribed to heteroatoms doping which could change the electron density of rGO. In the XRD pattern of GO (Figure S1C), there was a strong peak at around 11.0 °, which corresponded to an interlayer spacing of 0.825 nm for GO resulting from intercalation of oxygen-containing functional groups between graphene sheets.27 Whereas for the N,S-rGO, the peak at around 11.0 ° in XRD pattern was disappeared which was probably caused by partly oxygen-containing groups losing during the annealing process. Nevertheless, the N,S-rGO exhibited a broad peak at 26.1 ° corresponding to the (002) plane of graphite, which is consistent with above HRTEM result. Raman spectrum is employed to further confirm the existence of carbon. The N,S-rGO exhibited two prominent peaks at around 1350 cm-1 and 1587 cm-1 (Figure S2A, Supporting Information), which corresponded to



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disordered amorphous carbon (D-band) and graphitic sp2 carbon (G-band), respectively.28 The ID/IG value of N,S-rGO, which represented the defect intensity of graphene and the ratio of sp3/sp2 carbon,29 was around 1.07, further confirming the successful doping of the N and S heteroatoms.30-31 The FT-IR spectrum of the as-prepared GO (Figure S2B) provided the characteristic vibrations of the GO that included five major broad peaks at ca. 3440 cm-1, 1735 cm-1, 1633 cm-1, 1400 cm-1, and 1058 cm-1, which corresponded to the stretching vibrations of O-H group in hydroxyl, C=O in carbonyl groups, C=C, and the deformation peak of O-H and the epoxy band, respectively.32 However, in the IR spectrum of N,S-rGO (Figure S2B), the epoxy band at 1058 cm-1 disappeared, and that coincided with the reaction mechanism in which the N and S were co-doped by the catalyzed cyclization reaction. In addition, there was a new peak at ca. 1124 cm-1, which corresponded to C-N-C asymmetric stretching, conforming N atom was successfully doped. Meanwhile, the disappearance of the peak at 1735 cm-1 demonstrated that there was no C=O in the N,S-rGO, which was in agreement with the X-ray photoelectron spectroscopy (XPS) result below. By comparing the IR spectra of GO and N,S-rGO, GO was confirmed to be reduced to rGO after the doping process which was caused by the thermal treatment under inert atmosphere.33 All above results indicated that the as-prepared sample was N,S-rGO and the doping process didn't change the graphitic nature. The XPS full survey spectrum of N,S-rGO showed four peaks at 164, 284, 400, 533 eV, which corresponded to S 2p, C 1s, N 1s and O1s, respectively, definitely demonstrating the successful doping of N and S heteroatoms (Figure S3A).34 Meanwhile, the element content of C, N and S were 87.31%, 2.5% and 0.87%, respectively, which demonstrated that N,S-rGO was mainly based on carbon and doped with N and S. There were four distinguishable peaks presented in high-resolution C 1s XPS spectrum of N,S-rGO sample (Figure S3B), respectively representing sp2 C in graphene (284.1 eV); sp3 C in C-N, C-S (285.1 eV), and C-O (286.4 eV) and O-C=O (289.1 eV). While for GO, four obvious peaks presented in the high-resolution C 1s XPS spectrum are corresponded to the C-C, C-O, C=O and O-C=O, respectively (Figure S1D). In addition, the high-resolution N 1s spectrum of N,S-rGO (Figure 1C) showed two dominant peaks at 397.8 and 399.8 eV, corresponded to pyridine-like N and pyrrolic N.35 However, no peak at 400.9 eV was observed indicating that no graphitic N existed in N,S-rGO.35 Deconvolution of the S 2p peak illustrated the presence of S 2p3/2 and S 2p1/2 spin–orbit doublet (Figure 1D).36 The introduction of N and S atoms into graphene sheets


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was realized by catalyzing cyclization reaction between the carboxy groups, which existed on the surface of GO. Thus, 2-aminothiophenol played a significant role during the doping process. Compared to traditional doping method under high temperature, employing such a solvothermal method using 2-aminothiophenol as N and S source, we achieved in-situ N and S atoms co-doping simultaneously under lower temperature.37 3.2 N,S-rGO as an excellent quencher towards dual QDs-probe fluorescence GO is one of the widely used carbon materials which are ever adopted as a fluorescence quencher towards both organic dyes and QDs. However, the sp2 hybridized carbon property has imparted GO electronic conductivity to be further improved. Many efforts have been devoted to improve its electronic conductivity such as heteroatom doping. N, S are two frequently used elements for heteroatom doping.38 Here, N,S-rGO was prepared by one step procedure under mild condition and used as a fluorescence quencher towards QDs-probes. As shown in Figure 2A, when N,S-rGO was introduced into the 525 nm QDs-P1 solution, fluorescence intensity of the QDs-P1 gradually decreased as the concentration of N,S-rGO increased, ∆F was also investigated with the N,S-rGO concentration between 0.33 and 80.5 ng mL-1 (Figure 2B). Meanwhile, the fluorescence intensity of the QDs-P2 gradually decreased as the concentration of N,S-rGO increased when introducing N,S-rGO into the 623 nm QDs-P2 solution (Figure 2C), and ∆F was also investigated with the N,S-rGO concentration between 0.5 and 80 ng mL-1 (Figure 2D). 60 ng mL-1 N,S-rGO was used in the next experiments because the fluorescence intensity decrease approached to a platform beyond this concentration. The quenching efficiency is evaluated by recording the fluorescence decay time. The mean fluorescent decay time of QDs-P is 57 ns. In contrast, the average complex fluorescent decay time was 38 ns when quenched by bare GO and that was decreased to only 17 ns when quenched by N,S-rGO (Figure S4). The results were calculated by lifetime determine software with χ2 1.034, 1.068 and 1.092, respectively. These decay times have shown that the photo induced electron transfer between CdSe QDs and N,S-rGO is more effective than that between CdSe QDs and bare GO. 3.3 Feasibility of the virus DNA detection strategy and mismatched DNA determination In order to investigate the feasibility of the QDs-probes and N,S-rGO based “on-off-on” virus detection method, HBV and HIV DNAs were introduced into the QDs-probe and N,S-rGO mixed solution. The fluorescence intensity of QDs-P1 was recovered by adding HBV (Figure 3A), whereas that of QDs-P2 was recovered by adding HIV (Figure 3B). The fluorescence recovery demonstrated that the QDs-probe and N,S-rGO based method could be used to detect HBV and HIV DNA targets simultaneously. The selectivity of the dual viruses probes was investigated. As shown in Figure S5, for HBV



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detection, the fluorescence recovery intensity was decreased from one mismatched to three mismatched sequence. Meanwhile, there were the same results for HIV detection. It is demonstrated that the dual virus DNA detection method is highly selective towards their targets. Furthermore, the discrimination ability of the N,S-rGO based QDs probe toward single, double and triple mismatched DNA sequence may find widespread applications in detecting other analytes by altering the capture DNA sequences. 3.4 Dual virus DNAs determination by dual QDs-probes and N,S-rGO simultaneously and generality of this N,S-rGO based molecular sensing platform To investigate the sensitivity of the dual virus DNAs biosensor, the QDs probes were added into the virus DNA solutions. As shown in Figure 4, fluorescence intensity of QDs-P1 increased as the HBV concentration increased, and that of QDs-P2 also increased as the HIV concentration increased. The linear relationship for HBV detection is 5-100 nmol mL-1 with a detection limit of 2.4 nmol mL-1, and that for HIV detection is 7-80 nmol mL-1 with a detection limit of 3.0 nmol mL-1. Furthermore, only less than 5 min was required to finish the whole detection procedure. Obviously, this N,S-rGO based virus DNA detection method was sensitive, rapid and cost-effective. In addition, thrombin and IgE were chosen as protein analytes to evaluate the sensitivity and selectivity for verifying the generality of the N,S-rGO based molecular sensing platform. When altering the DNA probe loop domain to thrombin and IgE aptamers, the detection limits of as-prepared N,S-rGO based QDs probe were 0.17 ng mL-1 and 0.19 ng mL-1, respectively (Figure S6). The linear range was 0.4-4.2 ng mL-1 for thrombin, and that of 0.5-7.5 ng mL-1 was for IgE. It is clearly proved that this method is generous for sensing other biomolecules such as proteins only by altering the molecular beacon loop domain to target recognition element. 3.5 Detect the dual viruses DNA in the spiking samples It is necessary to study whether the N,S-rGO based QDs probe could be used in biological media. 10-40 nmol L-1 target viruses DNA were added into the 1% human serum samples to investigate the anti-interference ability of this virus detection method. As listed in Table 1, the quantitative recovery ranged from 99.0% to 100.6%, with a RSD less than 0.9%. This result proved that it was possible to realize detecting the targets virus in biological media by this N,S-rGO based detection platform with high sensitivity and rapid response time. 3.6 Kinetic characteristics of quenching reaction. The quenching efficiency of N,S-rGO towards QDs was investigated to verify the sensitivity and selectivity of the proposed sensor. The N,S-rGO was with excellent fluorescence quenching effect for the CdSe QDs after adding N,S-rGO into the QDs-probes solution (Figure S7, curve a, c, e, g). The QDs-probe fluorescence intensity was reduced more than 90% of the original one in less than 1 min. The strong and rapid quenching effect was due to the synergistic effect brought by N,


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S co-doping which could make the electron and energy transfer more efficient.14 However, the fluorescence intensity decrease of QDs-probe was not obvious when target DNA was mixed with QDs-Probe before adding N,S-rGO into the solution (Figure S7, curve b, d, f, h). The quenching effect of the N,S-rGO was significantly weakened because the existence of the target DNA could hinder the direct contact between N,S-rGO, whereas the fluorescence intensity could still maintain at least 67% of the original value. In addition, there was no detecting difference when altering the PBS buffer solution to the 1% human serum samples. This results proved that detecting virus DNA in biological media was possible by the N,S-rGO based detection platform.

4. CONCLUSION In summary, by using 2-aminothiophenol as N, S source through solvothermal approach, N,S-rGO has been synthesized and was further employed to quench CdSe QDs fluorescence by its excellent electronic transfer ability. It was combined as a biomolecular sensing platform for homogenous detection of virus DNA, proteins and any other analytes. Hairpin structured captured DNA sequence was designed by modified fluorescent QDs. When target analytes were recognized by captured DNA sequence, the interaction between them will induce the disclose between QDs and N,S-rGO as well as the fluorescence intensity recovery. Due to the unique co-doping structure and superior electronic transfer feature of N,S-rGO, sensitive and rapid detection results were obtained. In comparison with bare GO, N,S-rGO is a better candidate for rapid biological species detection. It provides a facile strategy for multiplex human being diseases related gene targets discrimination and detection.

SUPPORTING INFORMATION Morphology and structure characterizations of GO and N,S-rGO, such as TEM and SEM images, XRD pattern, XPS spectrum, Raman spectrum and FT-IR spectrum, additionally, PL decay curves, protein detection, fluorescence kinetic curves of quenching reaction. This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENTS Lu Chen and Liping Song contributed equally to this work. This work was supported by the National Natural Science Foundation of China (NSFC 21303064 and 21305049), Specialized Research Fund for the Doctoral Program of Higher Education of China (20130146120013), Wuhan Chenguang Science and Technology Project for Young Experts (2015070404010192), Fundamental Research Funds for the Central



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Universities, China (2662015PY163 and 2662015PY153), Natural Science Foundation of Hubei (2011CDC068) and the State Key Laboratory of Agricultural Microbiology of China (AMLKF201205).

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Scheme 1. Schematic illustration of the multi-colored QDs-P and N,S-rGO based HBV DNA and HIV DNA determination biosensor.

Figure 1. (A) TEM image of N,S-rGO and inset is its HRTEM image. (B) XRD pattern of N,S-rGO. (C) High-resolution N 1s and (D) High-resolution S2p XPS spectra of N,S-rGO.


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Figure 2. (A) Changes in the fluorescence spectra of 525 nm QDs-P1(1 nM) in 10 mM PBS solution (pH 8.0; λex = 388 nm) with an increasing concentration of N,S-rGO, with concentrations of 0, 0.33, 0.66, 0.99, 1.32, 2.64, 5.28, 10.5, 20.5, 40.5, 60.5, 80.5 ng mL−1 (from top curve to bottom curve). (B) The curve of quenched fluorescence intensity of 525 nm QDs-P1 upon increasing the N,S-GO concentration. (C) Changes in the fluorescence spectra of 623 nm QDs-P2 (1 nM) in 10 mM PBS solution (pH 8.0; λex = 388 nm) with an increasing concentration of N,S-GO, with concentrations of 0, 0.5, 1.0, 5.0, 10, 20, 40, 60, 80 ng mL−1 (from top curve to bottom curve). (D) The curve of quenched fluorescence intensity of 623 nm QDs-P2 upon increasing the N,S-rGO concentration.

Figure 3. (A) Fluorescence spectra of (a) QDs-P1; (b) QDs-P1 and N,S-rGO; (c) QDs-P1, N,S-rGO and HBV. (B) Fluorescence spectra of (a) QDs-P2; (b) QDs-P2 and N,S-rGO; (c) QDs-P2, N,S-rGO and HIV.



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Figure 4. (A) Fluorescence spectra of QDs-P and N,S-rGO; the concentrations of HBV DNA are 5, 10, 20, 60, 100 nM and that of HIV DNA are 7, 14, 35, 56, 80 nM (from bottom curve to top curve). (B) Linear curve for HBV DNA detection. (C) Linear curve for HIV DNA detection.

Table 1. Analytical results for the determination of HBV DNA and HIV DNA in human serum clinical samples. Sample

Added

Found

Mean

Mean

RSD

RSD

Recovery

Recovery for

for

for

for

HBV(%)

HIV(%)

HBV(%)

HIV(%)

(n=3)

(n=3)

1

8.0

8.0

7.92±0.03

8.05±0.06

99.0

100.6

0.81

0.57

2

16.0

16.0

15.89±0.05

16.08±0.05

99.0

100.5

0.35

0.26

3

32.0

32.0

32.21±0.08

31.92±0.04

100.6

99.8

0.60

0.48


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TOC figure:


Nitrogen and Sulfur Codoped Reduced Graphene Oxide as a General

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