AgNP-DNA@GQDs Hybrid: New Approach for Sensitive Detection of

Nov 12, 2014 - A growing body of evidence suggests that hydrogen peroxide (H2O2) plays an active role in the regulation of various physiological proce...
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AgNP-DNA@GQDs Hybrid: New Approach for Sensitive Detection of H2O2 and Glucose via Simultaneous AgNP Etching and DNA Cleavage Lili Wang,†,§ Jing Zheng,†,§ Yinhui Li,† Sheng Yang,† Changhui Liu,† Yue Xiao,† Jishan Li,† Zhong Cao,†,‡ and Ronghua Yang*,† †

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, and Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082, P. R. China ‡ Hunan Provincial Key Laboratory of Materials Protection for Electric Power and Transportation, School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha 410004, P. R. China S Supporting Information *

ABSTRACT: A growing body of evidence suggests that hydrogen peroxide (H2O2) plays an active role in the regulation of various physiological processes. Development of sensitive probes for H2O2 is an urgent work. In this study, we proposed a DNA-mediated silver nanoparticle and graphene quantum dot hybrid nanocomposite (AgNPDNA@GQDs) for sensitive fluorescent detection of H2O2. The sensing mechanism is based on the etching effect of H2O2 to AgNPs and the cleavage of DNA by as-generated hydroxyl radicals (•OH). The formation of AgNP-DNA@GQDs nanocomposite can result in fluorescence quenching of GQDs by AgNPs through the resonance energy transfer. Upon H2O2 addition, the energy transfer between AgNPs and GQDs mediated by DNA was weakened and obvious fluorescence recovery of GQDs could be observed. It is worth noting that the reaction product •OH between H2O2 and AgNPs could cleave the DNAbridge and result in the disassembly of AgNP-DNA@GQDs to achieve further signal enhancement. With optimal conditions, the approach achieves a low detection limit of 0.10 μM for H2O2. Moreover, this nanocomposite is further extended to the glucose sensing in human urine combining with glucose oxidase (GOx) for the oxidation of glucose and formation of H2O2. The glucose concentrations in human urine are detected with satisfactory recoveries of 94.6−98.8% which holds potential for ultrasensitive quantitative analysis of glucose and supplies valuable information for diabetes mellitus research and clinical diagnosis.

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graphene-metal hybrid material has attracted much attention in recent years due to their great promise in the field of biosensing,20−22 little attention has been paid to the GQDsmetal hybrid material.23 Hydrogen peroxide (H2O2) is a strategic attention of study into the molecular mechanisms underlying the development and progression of a disease.24 Given the low concentration and high reactivity of H2O2, developing a sensitive and selective sensing platform is one of the thought-provoking topics. In this paper, we attempt to utilize noncovalent interaction to construct a single strand DNA-mediated AgNP/GQDs hybrid nanocomposite and thus achieve high performance H2O2 detection. In this design, AgNPs acted as quencher and recognition unit.25,26 GQDs, which served as a signal output unit with excellent optical property, were assembled on the surface of DNA-modified AgNPs through π−π stacking. We demonstrate this DNAmediated self-assembled strategy can not only enhance the quenching efficiency but also improve the stability of AgNPs. Upon H2O2 addition, sensitive detection of H2O2 could be

anomaterials provide a promising sensing platform, because of the unique optical, electronic, and catalytic properties for translating the biorecognition events to an electrochemical or spectroscopic response.1−3 Plasmonic noble metal nanostructures, such as Ag and Au nanoparticle, have been intensively studied for a wide range of applications such as sensitive biosensing, nanomedicine, optoelectronics, and solar cells because of their unique tunable optical properties caused by localized surface plasmon resonance (LSPR).4−6 For instance, AgNPs have been successfully applied to realize colorimetric detection of various biomolecules with easy operation and low cost.7,8 However, limitations, such as low sensitivity, still existed. To address this issue, fluorescent strategies have attracted wide attention. Comparing with the colorimetric analysis, the fluorescent detection exhibits more advantages, including high sensitivity and selectivity and direct monitoring in live cells, tissues, and animals.9,10 Graphene quantum dots (GQDs) are zero-dimensional graphene sheets with lateral sizes less than 100 nm.11,12 Due to the property of photoluminescence contributed by quantum confinement and edge effects, GQDs have been quite attractive in optoelectronic devices, bioimaging, and photochemical catalysis. 13−19 Though it has been demonstrated that © XXXX American Chemical Society

Received: September 29, 2014 Accepted: November 12, 2014

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Preparation of AgNP-DNA@GQDs. Graphene oxide (GO) was synthesized from graphite powder according to Hummer’s method.30 GQDs were synthesized following the method reported by Tetsuka et al.31 In detail, 10 mL GO dispersion (0.1 mg mL−1) was mixed with 6 mL ammonia solution (25 wt % in water) and 10 mL deionized water in a glass vial. After stirring for 30 min, the mixture was transferred to a poly tetrafluoroethylene (Teflon)-lined autoclave (30 mL) and heated at 150 °C for 5 h. After cooling to room temperature, the mixture was filtered out using a 0.22 μm microporous membrane to separate insoluble large fragments. Then, the yellow filtrate was heated to 100 °C for 1 h to remove excess ammonia. The solution was further dialyzed in a dialysis bag (retained molecular weight: 8000 Da), and GQDs were in the reservoir outside the bag. The outside solution was then concentrated and further dialyzed through the dialysis bag (retained molecular weight: 1000 Da) for 2 days to remove the remaining tiny fragments. AgNPs were prepared by the reduction of AgNO3 with sodium borohydride in water.32 Thiol-DNA were modified on the surface of AgNPs according to the previous report.33,34 The number of DNA per AgNP was estimated by subtracting the amount of DNA in the supernatant mixture from the total amount of DNA, which was added into the AgNPs solution. Then, the AgNP-DNA was redispersed in BR buffer and a final concentration of 10 μg/mL GQDs were added; the solution was incubated at room temperature for 2 h to prepare AgNP-DNA@GQDs. The final concentration of AgNP-DNA@GQDs was defined as the concentration of AgNPs and calculated using Lambert−Beer’s law according to the following equation: A = εbc, where the extinction coefficient (ε) is 8.47 × 109 M−1 cm−1 for 15 nm AgNPs and b = 1 cm (for standard cuvettes).22,35,36 Fluorescence Detection of H2O2. To detect H2O2, aliquots (400 μL) of BR buffer (pH 5.1) containing 150 pM AgNP-DNA@GQDs were spiked with H2O2 (0−500 μM) and reacted at 37 °C for 35 min. Then, the fluorescence was measured under excitation at 340 nm. Glucose Sensing. In a typical experiment, 380 μL aliquots of the as-prepared AgNP-DNA@GQDs suspension containing GOx (0.5 mg mL−1) were incubated with 20 μL glucose solutions of varied concentration (0−2000 μM) at 37 °C for 40 min. For fluorescent detection of glucose in urine, three urine samples from one diabetic patient and one healthy volunteer before and after 50 g oral glucose tolerant test were collected. The urine (20 μL) was first diluted to 200 μL using BR buffer containing 100 μM Zn2+ for the measurements, and then 20 μL diluted urine sample was added into 380 μL as-prepared AgNPDNA@GQDs suspension containing GOx and incubated at 37 °C for 40 min. The fluorescence was measured under excitation at 340 nm.

achieved via simultaneous AgNPs etching and DNA cleavage. As further application, glucose in urine is detected and the sensing assay is mainly based on the enzymatic conversion of glucose by glucose oxidase (GOx) to form H2O2.27 Compared with the known fluorescence-based H 2 O 2 detection strategies,28,29 our proposed AgNP-DNA@GQDs hybrid nanocomposite possesses some remarkable features: first, as the fluorescence report element, the long-term photo stability of GQDs can ensure ideal signal output; second, the effective link agent DNA play important roles in our design. On one hand, the self-assembly between DNA and GQDs based on π−π stacking interactions can not only preserve the optical properties of GQDs well but also shorten the distance between GQDs and AgNPs and thus effectively quench the fluorescence and reduce the background before H2O2 addition. Most importantly, the DNA can be cleaved by hydroxyl radical (•OH) produced by the reaction between AgNPs and H2O2. This cleavage can further disassemble AgNP-DNA@GQDs and thus achieve H2O2 detection at lower concentration. Third, this strategy is generalizable and multiple H2O2-involved analytes detection such as glucose can be realized, which allows for facile detection of various targets without increasing the complexity and cost of the nanocomposite. Therefore, this approach is not only sensitive and selective but also convenient and generalizable.

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EXPERIMENTAL SECTION Chemicals and Reagents. Oligonucleotides and dialysis bags (molecular weight cut off =1000 and 8000) used in this work were obtained from Sangon Biological Technology & Services Co., Ltd. (Shanghai, China). High purity graphite powder was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Tris(2-carboxyethyl)phosphine (TCEP), allopurinol, and glucose oxidase (GOx) were purchased from Sigma-Aldrich. 40 mM Britton-Robinson (BR) buffer was prepared by dissolving of H3BO3, H3PO4, and CH3COOH in distilled water, and the final pH value is adjusted by 0.2 M NaOH. All solutions were prepared and diluted using ultrapure water (sterile Millipore water, 18.2 MΩ). All other reagents were analytical reagent grade and were used without further purification or treatment. Instruments and Characterization. Transmission electron microscopy (TEM) was conducted on a JEOL (JEM 3010) electron microscope at an acceleration voltage of 200 kV and high-resolution transmission electron microscopy (HRTEM) images were collected on a Tecnai G2 F20 STWIN electron microscope (FEI Company), using a 200 kV accelerating voltage. Atomic force microscopy (AFM) images were performed on a SPI3800N-SPA400 (Seiko Instruments, Inc.). X-ray photoelectron spectroscopy (XPS) data were obtained by an ESCA Lab electron spectrometer (Thermal Fisher Scientific) using 300 W Al Kα radiation. The base pressure was about 3 × 10−9 m bar. The binding energies were referenced to the C 1s line at 284.8 eV from adventitious carbon. Curve fitting of the C 1s and N 1s spectra was performed using a Gaussian−Lorentzian peak shape. UV−vis absorption spectra were obtained on a Hitachi U-4100 UV−vis spectrophotometer (Kyoto, Japan). The fluorescence emission spectra and fluorescence anisotropy were obtained on a PTI QM4 Fluorescence System (Photo Technology International, Birmingham, NJ). The pH was measured by a model 868 pH meter (Orion).

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RESULTS AND DISCUSSION Sensing Scheme. As shown in Scheme 1, a single strand DNA (5′-SH-AAAGAAAGAAAGAAAGAAAG-3′) was labeled with sulfhydryl and then conjugated to the surface of AgNPs through the Ag−S bond. The subsequent π−π stacking interactions between DNA and GQDs can achieve successful assembly of AgNP-DNA@GQDs. In the absence of H2O2, AgNPs displayed extremely high quenching efficiency, and the fluorescence of GQDs were quenched due to the overlap between the emission spectrum of as-prepared GQDs (420 nm) and the absorption spectrum of AgNP-DNA (400 nm), which can satisfy the principle of resonance energy transfer B

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calculated using Lambert−Beer’s law. Then, the single strand DNA was conjugated to the surface of AgNPs using the Ag−S bond. Because of the physisorption ability between DNA nucleobases and graphene following the order of G > A > T > C, the DNA sequence rich in G and A nucleobase was selected to enhance the absorption of GQDs.46 After conjugated with DNA, the UV−vis spectrum of AgNP-DNA exhibit a strong absorption centered at 400 nm and about 6 nm red shift compared with bare AgNPs (Figure S5 of the Supporting Information), which is attributed to the decrease of confinement in one dimension. Due to the strong π−π stacking, the GQDs can assemble onto the surface of AgNP-DNA and then result in formation of a hybrid AgNP-DNA@GQDs nanocomposite. We can observe satellites assembly formed with multiple GQDs surrounding AgNPs in TEM images, as shown in Figure 1A. For the control experiment, we mixed the bare

Scheme 1. Schematic Description of H2O2 and Glucose Detection Based on AgNP-DNA@GQDs

(RET) in our experiment.22,37 Upon H2O2 addition, AgNPs could be etched effectively, and the decreased absorbance of AgNPs can thus result in the fluorescence recovery of GQDs. Meanwhile, DNA-bridge was cleavaged by hydroxyl radical (•OH),38,39 which was generated from the Fenton-like reactions between H2O2 and AgNPs.40−43 In this case, GQDs were far away from the surface of AgNP-DNA and the releasing AgNPs were aggregated together in salt-containing BR buffer. Since the intensity of fluorescence recovery of GQDs was proportional to the concentration of H2O2, therefore, fluorescent H2O2 and glucose detection can be achieved. Characterization of AgNP-DNA@GQDs Nanocomposite. Hybrid AgNP-DNA@GQDs nanocomposite was synthesized via a multistep procedure, which involved GQDs synthesis, AgNP-DNA conjugation, and final assembly of GQDs onto the AgNP-DNA surfaces. As a signal report element, the morphology of the obtained GQDs was first characterized using TEM, as shown in Figure S1A of the Supporting Information. The result shows a size distribution from 2 to 5 nm, and the average diameter was about 3.3 nm. The HRTEM image (see inset of Figure S1A of the Supporting Information) clearly shows the lattice spacing of 0.22 nm, which is consistent with the (102) diffraction planes of sp2 graphitic carbon.44 Then, the AFM image (Figure S1B of the Supporting Information) shows the topographic height of the obtained GQDs, which mostly distributed in the range from 0.5 to 1.3 nm, with an average value of 0.9 nm (inset of Figure S1B of the Supporting Information), suggesting that most GQDs consist of a single graphene layer. To further characterize the composition of GQDs, XPS was performed. The full scan XPS spectrum of the GQDs (Figure S2A of the Supporting Information) shows the existence of carbon (C 1s, 285 eV), nitrogen (N 1s, 400 eV), and oxygen (O 1s, 533 eV). The high resolution scan of the C 1s, N 1s, and O 1s (Figure S2B−S2D of the Supporting Information) confirm the graphitic structure of GQDs, and the amino-groups might be preponderantly located in the edges of graphene layers.31,45 Next, we investigated the optical properties of GQDs. Upon excitation with a 340 nm beam, the fluorescence spectrum of GQDs shows a strong peak at 420 nm. The fluorescence quantum yield (QY) is 17.6%, which is calibrated against quinine sulfate as a standard sample (54% in 0.5 M H2SO4). The GQDs are photostable and remain almost invariant even under continuous irradiation (365 nm) with a Xe lamp for 12 h (Figure S3 of the Supporting Information). As for the key constituent of this constructed nanocomposite, AgNPs were synthesized according to the previous report and characterized by TEM. The asprepared AgNPs are well-monodispersed, and the sizes are about 15 nm (Figure S4 of the Supporting Information). The final concentration of AgNPs is about 1.6 nM, which is

Figure 1. TEM image of AgNP-DNA@GQDs (A) before and (B) after 200 μM H2O2 addition. The GQDs were indicated with the arrows.

AgNPs with GQDs directly, and AgNPs and GQDs were distributed randomly, as shown in Figure S6 of the Supporting Information. To further confirm the successful fabrication of AgNP-DNA@GQDs nanocomposite, fluorescence anisotropy (FA), a simple and robust signaling transduction approach which gives information on molecular mobility was employed.47 As shown in Figure S7 of the Supporting Information, the FA value of the free state of GQDs in buffer solution is very low, and a significant anisotropy increase could be observed upon addition of AgNP-DNA. In contrast, the FA increment is much weaker upon addition of bare AgNPs. All these results could indicate the successful fabrication of AgNP-DNA@GQDs nanocomposite. Figure S8A of the Supporting Information shows there is a large overlap between emission spectrum of GQDs and absorption spectrum of AgNP-DNA. After GQDs were assembled onto the surface of AgNPs using single strand DNA, as shown in Figure S8B of the Supporting Information, significant fluorescence decrease of GQDs could be observed, indicating that effective RET occurs between GQDs and AgNPDNA. The fluorescence of GQDs was decreased rapidly upon addition of an increasing amount of AgNP-DNA, and the results showed that more than 94% quenching was achieved upon 150 pM AgNP-DNA addition. Meanwhile, to verify that single strand DNA can effectively mediate the assembly of AgNPs and GQDs and thus enhance RET between these two nanoparticles, we also investigated the fluorescence quench of GQDs upon bare AgNPs addition. The result demonstrates that the fluorescence quenching of GQDs is relatively weaker than that of AgNP-DNA addition (inset of Figure S8B of the C

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enhancement (F/F0) reached the maximum, where F and F0 are the fluorescence intensities in the presence or absence of H2O2. Therefore, 30 mM NaNO3 was chosen as the optimal concentration in the subsequent experiment. Considering the lower pH of buffer solution is beneficial to the Fenton-like reaction between AgNPs and H2O2,40−42 we next studied the response of our constructed AgNP-DNA@GQDs upon H2O2 addition in different pH buffer solution (fluorescence spectrum were shown in Figure S13 of the Supporting Information). As shown in Figure S14 of the Supporting Information, pH 5.1 was selected in the subsequent experiments. Under optimized condition, Figure 3A shows the fluorescent spectra of AgNP-DNA@GQDs in the presence of variable

Supporting Information), which further imply the feasibility of our constructed “switch-on” fluorescent sensing system with a lower background signal. Fluorescent Detection of H2O2. The underlying chemistry involved in our design is based on etching AgNPs from the hybrid AgNP-DNA@GQDs nanocomposite using H2O2. The etching may go through a process similar to the Fenton reaction:40 Ag + H 2O2 + H+ = Ag + + •OH + H 2O

First, we investigated the morphology of this nanocomposite upon H2O2 addition using TEM. As shown in Figure 1B, GQDs were far away from AgNP-DNA due to the removal of DNA from AgNPs surface by H2O2 and generated •OH. In this case, salt-induced AgNPs aggregation occurred. Next, the typical UV−vis spectral response of AgNP-DNA@GQDs before and after incubation with H2O2 is shown in Figure 2A.

Figure 3. (A) Fluorescence spectra of AgNP-DNA@GQDs as functions of different concentrations of H2O2. The arrow indicates signal changes as H2O2 concentrations increase (0.4−500 μM). (B) Fluorescence enhancement (F/F0) of AgNP-GQDs (curve a) and AgNP-DNA@GQDs (curve b) at 420 nm on the increasing concentrations of H2O2, where F and F0 are the fluorescence intensities of system in the presence or absence of H2O2. The error bars signify the standard error obtained from three repetitive measurements.

Figure 2. (A) UV−vis absorption of (a) AgNP-DNA, (b) AgNPDNA@GQDs, and (c) AgNP-DNA@GQDs upon 200 μM H2O2 addition. (B) Fluorescence emission spectra of (a) GQDs, (b) AgNPDNA@GQDs, and (c) AgNP-DNA@GQDs upon 200 μM H2O2 addition. The spectra were collected in the BR buffer (pH 5.1) containing 30 mM NaNO3. λex = 340 nm.

concentrations of H2O2. The AgNP-DNA@GQDs nanocomposite exhibited very weak fluorescence in the absence of H2O2. However, the fluorescence intensity around 420 nm gradually enhanced upon increasing H2O2 addition. The calibration curves were also established by plotting the increment of fluorescence at 420 nm versus H2O2 concentration (curve a, Figure 3B). A good linearity concentration was raised from 0.4 to 200 μM, deriving a detection limit of 0.10 μM. Meanwhile, a control experiment, in which the bare AgNPs and GQDs were mixed together for H2O2 detection was employed. The result showed that the fluorescence of GQDs was quenched by bare AgNPs through the inner filter effect (IFE) to some degree which was mainly due to the complementary overlap region between the absorption spectrum of AgNPs and the emission spectrum of GQDs.48,49 Upon H2O2 addition, AgNPs were etched, and the decreased absorbance of AgNPs caused the IFE to be less effective. Thus, measurable fluorescence recovery of GQDs could be observed. However, as shown in Figure 3B, the result reveals that this response is not as sensitive as our constructed AgNP-DNA@ GQDs nanocomposite. The detection limit of AgNP-DNA@ GQDs was obviously lower than bare AgNPs and GQDs mixture system for the detection of H2O2 (0.39 μM, curve b, Figure 3B). The limit of detection (LOD) was calculated with the following equation: LOD = 3σ/k, where σ is the standard deviation for the blank solution of ten measurements and k is the slope of the calibration curve. The results indicate that our constructed AgNP-DNA@GQDs nanocomposite is appropriate for highly sensitive quantification of H2O2 in aqueous solution.

As expected, it was evident that a drastic absorption change could be observed, which further confirmed that AgNPs were etched and aggregated upon H2O2 addition. Subsequently, we investigated the effect of H2O2 to the fluorescence of GQDs, and the result shows that the fluorescence intensity is invariable as a variation of H2O2 concentration (Figure S9 of the Supporting Information). Our experiments further indicated that obvious fluorescence recovery for the hybrid AgNPDNA@GQDs nanocomposite was observed upon H 2O2 addition (Figure 2B). The time-dependent fluorescence spectrum revealed the reaction achieved equilibrium in 35 min with the maximum intensity (Figure S10 of the Supporting Information). These results confirm the feasibility of our constructed AgNP-DNA@GQDs of fluorescent H2O2 detection. Since the DNA number conjugated on the surface of AgNPs can influence the signal response to H2O2, we next optimized the ratio of DNA to AgNP. The maximum response (F/F0) was obtained with a loading capacity of about 68 DNA per AgNP (Figure S11 of the Supporting Information). However, higher concentration could cause the decrease of F/F0, which is due to the steric hindrance of too dense DNA. Owing to the possibility of higher salt concentration causing the aggregation of AgNPs and thus influencing the response of our constructed AgNP-DNA@GQDs upon H2O2 addition, we next investigated the effect of NaNO3 on our system. As shown in Figure S12 of the Supporting Information, when the concentration of NaNO3 contained in the BR buffer equaled 30 mM, the fluorescence D

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Effect of Generated •OH. It has been reported that the reaction between AgNPs and H2O2 was similar to the Fenton reaction, which could produce powerful oxidizing species such as •OH. The as-generated •OH was proved to cleave the DNA phosphate backbone in a largely sequence-independent manner. Therefore, one of the most important features of our design is that the single strand DNA can be cleaved by •OH and thus further disassemble AgNP-DNA@GQDs nanocomposite to achieve signal amplification upon H2O2 addition. First, the cleavage of as-generated •OH to fluorescein (FAM)-labeled DNA was investigated by agarose gel electrophoresis images (details were shown in the Supporting Information). As shown in Figure S15 of the Supporting Information, there was little effect to the single strand DNA sequences by H2O2 or AgNPs alone (lane b and c), however, obvious weak band was obtained on the gel when there is coexistence of AgNPs and H2O2 (lane d). Therefore, comparing all the bands in the image, we could conclude that the DNA breakage occurred by the as-generated •OH. Then, to confirm the synergistic effect of the •OH-induced signal amplification, allopurinol, a typical •OH-scavenger, was used.50 The kinetic behaviors of AgNP-DNA@GQDs as well as their response to H2O2 before and after allopurinol addition were then studied. The fluorescence intensity of AgNP-DNA@ GQDs gradually increased as a function of time upon treatment with H2O2. As shown in Figure 4, the increasing trend of the

catalyze the oxidation of glucose in the presence of oxygen to form H2O2:25,51 D − glucose + O2 + H 2O GOx

⎯⎯⎯→ D − gluconic acid + H 2O2

By combining this reaction with the AgNP-DNA@GQDs nanocomposite, high sensitive detection of glucose is feasible based on the fluorescence enhancement associated with the produced H2O2. In our design, since the catalytic activity of GOx is pH-dependent,52 we first investigated the effect of pH on our constructed AgNP-DNA@GQDs used for glucose detection (fluorescence spectrum shown in Figure S16 of the Supporting Information). It can be concluded from Figure S17 of the Supporting Information, that a weak acid environment (around pH 5.6) is suitable to attain maximum significant fluorescence enhancement upon glucose addition. After optimizing the experimental parameters, we measured the fluorescence emission spectra of AgNP-DNA@GQDs in the BR buffer containing glucose at varying concentrations (Figure 5A). A weak emission peak was demonstrated before glucose

Figure 5. (A) Fluorescence spectra of AgNP-DNA@GQDs as functions of different concentrations of glucose. The upward arrow indicates the signal changes with the increases in glucose concentration (2.0−2000 μM). Inset: plot of F/F0 versus glucose concentration (2.0−100 μM), where F and F0 are the fluorescence intensities of system in the presence or absence of glucose. (B) Fluorescence response of the AgNP-DNA@GQDs to glucose (500 μM) or competing carbohydrates (1 mM) and the mixture of the competing carbohydrates with glucose in buffer solution. The error bars signify the standard error obtained from three repetitive measurements.

Figure 4. Time-dependent fluorescence change of AgNP-DNA@ GQDs in the absence (curve a) and presence of 10 mM allopurinol (curve b) upon addition of 200 μM H2O2. Inset: Effect of allopurinol with different concentrations on the kinetic plots of I[(Fmax − Ft)/ Fmax] vs time for the reaction of AgNP-DNA@GQDs with H2O2. Fluorescence was monitored at 420 nm with an excitation wavelength of 340 nm.

addition, while significant emission enhanced at 420 nm was observed upon the addition of glucose to the solution. The fluorescence intensity increased in proportion to the amount of glucose in the solution (i.e., from 2.0 to 2000 μM). Meanwhile, a linear calibration graph is achieved by plotting the fluorescence increment versus glucose concentration within 2.0 to 100 μM, deriving a detection limit of 0.42 μM (inset, Figure 5A). Table S1 of the Supporting Information compares the analytical performance of the present glucose sensing system with some of the reported procedures for glucose detection. The AgNP-DNA@GQDs hybrid system is more sensitive than the reported approaches. Figure 5B shows the response of AgNP-DNA@GQDs hybrid toward glucose (500 μM) and some other carbohydrates (1 mM). The results showed that other carbohydrates (fructose, galactose, mannose, matose, lactose, sucrose, and xylose) could not induce any obvious change and thus have little interference for glucose sensing. Additionally, a titration of glucose in the presence of the interfering carbohydrates is almost superimposable on the one obtained exclusively in the presence of glucose, which

fluorescence intensity of AgNP-DNA@GQDs after H2O2 introduced was obviously slower upon allopurinol (10 mM) addition. The time-dependent processes of AgNP-DNA@ GQDs response to H2O2 with different concentrations of allopurinol followed pseudo-first-order kinetics with a different observed rate constant k′ (Figure 4, inset), and the relevant observed rate constants of AgNP-DNA@GQDs with higher concentrations of allopurinol were slower. Meanwhile, the observed rate constant of AgNP-DNA@GQDs (k′AgNP‑DNA@GQDs = 1.52 × 10−3 s−1) was much larger than that of 10 mM allopurinol addition (k′AgNP‑DNA@GQDs+allopurinol = 8.9 × 10−4 s−1). Therefore, the results further confirmed that as-generated •OH could actually accelerate the reaction rate and achieve signal amplification for sensing H2O2 sensing. Fluorescent Detection of Glucose. The successful sensitive detection of H2O2 was then implemented for the analysis of glucose. It is known that GOx can specifically E

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indicates that the AgNP-DNA@GQDs-based detection system exhibits a specific response to glucose due to the substrate specificity of GOx. This provides an excellent selectivity in the fabrication of the glucose biosensor based on the AgNP-DNA@ GQDs nanocomposite. Preliminary Application. The practical applicability of the present method was tested on the assay of glucose detection in human urine. The glucose level in urine can be used as an indication of worsening of diabetes.53,54 Since human urine would contain biologically related metals, anions, or molecules, we first added 100 μM Zn2+ in buffer to eliminate the interference of urea and uric acid that can absorb on the surface of AgNPs and protect them from etching.8 Following the described procedure, the response of the nanocomposite to glucose in the presence of the interfering species in urine was investigated by addition of different amounts of the interfering agent to the samples containing 500 μM glucose. As shown in Table S2 of the Supporting Information, the substances existing in urine showed no remarkable interference for the determination after 200 times dilution. To test the applicability of the proposed assay, glucose was spiked into one urine sample with diabetes, two samples from health volunteer before and after 2 h for taking 50 g oral glucose tolerant test. The level of glucose was detected by the present method and glucometer, and the experimental results were shown in Table S3 of the Supporting Information. The results provided by the proposed method are close to the values determined by the glucometer which is in accordance with the fact that the glucose concentration in healthy human urine sample is below 1 mM.55 Glucose concentration recoveries of 94.6−98.8% were achieved, and the relative errors were no more than 6%. These results indicate that the proposed method can be used in the detection of glucose in urine.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 86-731-88822523. Fax: 86731-88822523. Author Contributions §

L.W. and J.Z. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for the financial support through the National Natural Science Foundation of China (Grants 21305036, 21135001, 21405038, 21475036, and J1103312), the Foundation for Innovative Research Groups of NSFC (21221003), the “973” National Key Basic Research Program (2011CB911000), and the Fundamental Research Funds for the Central Universities.

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CONCLUSION In summary, a new DNA-mediated hybrid AgNP-DNA@ GQDs fluorescent nanoplatform for H2O2 detection is proposed in this paper. By taking advantage of the strong π−π stacking interactions between DNA and GQDs and the etching effect of H2O2 to AgNPs, this assay is simple, convenient, and sensitive. Meanwhile, high sensitivity was an achieved benefit from the DNA-mediated low background and the as-generated •OH. The new DNA-mediated hybrid AgNPDNA@GQDs fluorescent nanoplatform could also be easily applied for other substrate detection, involving the generation of H2O2, such as choline (catalyzed with choline oxidase), lactic acid (catalyzed with lactate oxidase), and so on.56,57 As further application, glucose detection in human urine is achieved combining with GOx, which holds potential for ultrasensitive quantitative analysis of glucose and supplies valuable information for diabetes mellitus research and clinical diagnosis. Therefore, we expect that this strategy may offer a new approach for developing low cost and sensitive sensors for biological and clinical diagnosis applications.

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ASSOCIATED CONTENT

* Supporting Information S

More experimental details and spectroscopic data as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. F

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