Disaggregation Behavior of Graphene

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Tuning the Aggregation/Disaggregation Behavior of Graphene Quantum Dots by Structure-Switching Aptamer for High-Sensitivity Fluorescent Ochratoxin A Sensor Song Wang, Yajun Zhang, Guangsheng Pang, Yingwei Zhang, and Shaojun Guo Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03913 • Publication Date (Web): 28 Dec 2016 Downloaded from http://pubs.acs.org on December 28, 2016

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Analytical Chemistry

Tuning the Aggregation/ Aggregation/Disaggregation Behavior of Graphene Quantum Dots by Structuretructure-Switching witching Aptamer for HighHigh-Sensitivity Fluorescent Ochratoxin A Sensor Song Wang,† Yajun Zhang,† Guangsheng Pang,§ Yingwei Zhang*† and Shaojun Guo*‡# †

State Key Laboratory of Chemical Resource Engineering & Key Laboratory of Carbon Fiber and Functional Polymers, Ministry of Education; College of Materials Science & Engineering, Beijing University of Chemical Technology, Beijing, 100029, China ‡

Department of Materials Science and Engineering, College of Engineering, Peking University, Beijing 100871, China.

#

BIC-ESAT, College of Engineering, Peking University, Beijing 100871, China.

§

State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, College of Chemistry, Jilin University, Changchun, 130012, China Keywords: graphene quantum dots; Ochratoxin A; structure-switching; aptasensor; fluorescent sensors ABSTRACT: The design of graphene quantum dots (GQDs)-aptamer bioconjugates as the new sensing platform is very important for developing high-sensitivity fluorescent biosensors, however achieving new bioconjugates is still a great challenge. Herein, we report the development of a new high-sensitivity fluorescent aptasensor for the detection of ochratoxin A (OTA) based on tuning aggregation/disaggregation behavior of GQDs by structure-switching aptamers. The fluorescence sensing process for OTA detection involved two key steps: 1) cDNA-aptamer (cDNA: complementary to part of the OTA aptamer) hybridization induced the aggregation of GQD (fluorescence quenching) after cDNA was added into the GQDs-aptamer bioconjugate solution and, 2) the target of OTA triggered disaggregation of GQD aggregates (fluorescence recovery). Such new fluorescent sensing platform can be used to monitor OTA with a linear range of 0 to 1 ng/mL and very low detection limit of 13 pg/mL, which is among the best in all the developed fluorescent nanoparticlesbased sensors. Such sensing strategy is also successful in analyzing OTA in practical red wine sample with 94.4-102.7% of recoveries and relative standard deviation in the range of 2.9-5.8%. The present works open a new way for signaling the target-aptamer binding event by tuning aggregation/disaggregation behavior of GQDs-bioconjugates.

The ochratoxin A (OTA), a type of mycotoxin existing in a wide variety of foods, is one of most interesting bioanalytes due to its high toxicity to the hepar and kidney of human and potential carcinogenic hazard.1,2 Currently, the typical methods for the detection of OTA rely on high-performance liquid chromatography (HPLC),3,4 mass spectrometry (MS),5 and immunoassay.6-9 However, these conventional detecting methods using HPLC or MS require expensive equipment and timeconsuming sample pretreatment. Furthermore, the immunoassay using antibody molecules as recognition element for OTA detection, such as enzyme linked

immunosorbent assay (ELISA)6,7 electrochemical 8 immunosensors and array immunosensors,9 suffers from the high cost, cross reactivity and poor stability of antibody molecules. Developing a simple, rapid, sensitive and low cost detection method for high-sensitivity detection of OTA is very necessary for ensuring the food safety. Aptamer is single stranded DNA or RNA molecule with the special ability for binding a variety of targets for developing multiple biosensors.10 Since the discovery of the aptamer specific for OTA,11 aptasensors have been widely constructed for detecting OTA using

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colorimetric,12 fluorescent,13 electrochemical,14 surfaceenhanced Raman scattering sensing platforms.15 Among

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them, fluorescent aptasensors are of more interest due to

Scheme 1. Schematic illustration on the new fluorescent signal-on sensing platform for OTA detection based on the aggregation state change of GQDs from dispersion to aggregation to disaggregation due to the aptamer structure switching from single strand to aptamer/cDNA duplex to aptamer/OTA complex.

their advantages in high sensitivity and convenience. Compared with using organic fluorescent dyes as label for fluorescent aptasensors, upconversion fluorescence nanoparticles and semiconductor quantum dots show the more obvious advantages in developing stable and highsensitivity aptasensors,16,17 but achieving aptamerinorganic fluorescent nanoparticle bioconjugates for developing high-sensitivity aptasenosrs is still a great challenge. The recent emergence of fluorescent graphene quantum dots (GQDs) has sparked intensive research interests due to its excellent photostability, hydrophilic, good biocompatibility and low toxicity.18,19 One of interesting properties of GQDs is that their fluorescence can be easily switched on and off through controlling the disaggregation/aggregation state of GQDs. By further considering the structure-switching signaling aptamers are a special class of aptamer reporters, which can report the target binding event by switching structure from DNA duplex to DNA/target complex, herein, we proposed a simple fluorescence signal-on strategy for the highsensitivity OTA detection by applying GQDs as the fluorescent probe, based on the aggregation state change of GQDs from aggregation to disaggregation induced by the aptamer structure switching from aptamer-cDNA (a designed short complementary DNA) duplex to aptamertarget complex. Scheme 1 shows the schematic illustration on sensing OTA based on the aptamer structure switching trigged disaggregation of GQDs aggregates and subsequent fluorescent enhancement. To be specific, GQDs were first modified on the 3’-end of aptamer strand and the 5’-end of cDNA strand, forming GQDs-DNA conjugates (named as GQDs-aptamer and GQDs-cDNA, respectively). GQDs-aptamer and GQDs-

cDNA with identical amounts were mixed together, and consequently, GQDs were brought into proximity as a result of the hybridization of aptamer/cDNA, leading to the quenching of GQDs fluorescence. Finally, the addition of target OTA would induce the structure switching from aptamer/cDNA duplex to aptamer/OTA complex, resulting in the disaggregation of GQDs with enhanced fluorescence. Through this strategy, we can perform the fluorescent “signal-on” detection of OTA with higher sensitivity and lower detection limit compared with the reported fluorescent biosensors,20-22 and specially realize the successful evaluation of OTA concentration in practical red wine sample. To the best of our knowledge, this is the first report on combining GQDs with switchable fluorescence controlled by aggregation/disaggregation with structure-switching signaling aptamer strategy to reveal the target-aptamer binding event, which can be further extended to other biodetection system to construct fluorescent switch-on aptasensor. EXPERIMENTAL SECTION Reagents and apparatus. OTA, (ochratoxin B) OTB, (aflatoxin B1) AFB1 and Zearalenone (ZEN) were obtained from Sigma-Aldrich. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), Nhydroxysuccinimide (NHS) were purchased from Shanghai Aobo Biotechnology Co. Ltd. All the other chemicals were purchased from Beijing Chemical Reagent Co. Ltd. All chemicals were used without further purification and the water used throughout all experiments was purified using a Millipore system. The buffer solutions used in all the experiments were prepared


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as follow: PBS buffer (0.1 M Na2HPO4/NaH2PO4, pH=7.4, 100 mM NaCl), TE buffer (40 mM Tris, 2 mM EDTA, pH=7.4). The DNA oligonucleotides were purchased from Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China), and their base sequences are given as follows:23,24 cDNA: (complementary to part of the OTA aptamer) 5’-NH2-C6H12-TGTCCGATGCT-3’; OTA aptamer: (the complementary base sequences with cDNA are underlined) 5’-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA-C6H12-NH2-3’. Synthesis of graphene oxide (GO). GO was prepared from natural graphite powder through a modified Hummers method.25 First, 4.6 mL of 98% H2SO4 was added into a round-bottomed flask located in ice-bath. Then, 0.2 g graphite powder and 0.1 g NaNO3 were carefully added into the flask under continuous stirring of 15 min in ice-bath, followed by an extremely slow addition of 0.6 g KMnO4. Next, the temperature was raised to 35 oC and kept for 30 min at this temperature. 9.2mL of distilled water was added into the above system and the temperature was further increased to 98 oC, keeping stirring for 30min. Finally, a mixture of 28 mL distilled water and 2 mL of 30% H2O2 was added into the system for stopping the reaction. The resulting solution was filtered, and the collected products were washed by distilled water until the pH value of filtered solution is neutral. The products were dried under vacuum and freeze temperature. Synthesis of graphene quantum dots (GQDs). 50 mg GO were oxidized in 98% H2SO4 and 68% HNO3 with a ratio of 3:1 for 2 h under mild ultrasonication (500 W, 40 kHz), followed by dilution treatment of distilled water. Then, the oxidized products were filtered using a 0.22-μm microporous membrane and re-dispersed in 40 mL of distilled water. The suspension was transferred to an autoclave (50 mL) and heated at 180 oC for 12 h. After cooling to room temperature naturally, the black suspension was filtered through a 0.22-μm microporous membrane, and the brown filtered solution was retained. The obtained filtered solution was further dialyzed in a dialysis bag (retained molecular weight: 3500 Da) for 3 days. The GQDs powder was finally obtained after freezedrying treatment. Preparation of GQDs-DNA probe. GQDs were first dissolved in 10 mL PBS buffer to obtained GQDs suspension (0.1 mg/mL), followed by adjusting pH value to 5 for protonation of carboxyl groups of GQDs. After that, a mixture of EDC (19 mg) and NHS (22 mg) was added into the solution to activate carboxylic group of GQDs under stirring treatment of 30 min at room temperature. Then, GQDs suspension was divided to two equal parts, each 5 mL in flask, after adjusting pH value to 7.4. The GQDs-aptamer conjugates were obtained by adding 24 µL of amino-modified aptamer (100 µM) into

one of the above activated solution to conduct the condensation reaction for 2 h at room temperature with continuous stirring. In a similar way, we obtained GQDscDNA conjugates. All of these GQDs-DNA conjugates were all centrifuged using centrifugal ultrafiltration tubes with a speed of 8000 rmp for 10 minutes and washed with PBS buffer three times to remove excessive DNA before finally dispersing them in PBS buffer and storing at 4 oC. Fluorescence detection experiments. The fluorescence quenching experiment of GQDs-aptamer and GQDs-cDNA was first performed as follow: 500 μL of GQDs-aptamer suspension was mixed with the same volume of GQDs-cDNA suspension at room temperature under gentle shaking, and the fluorescence quenching of this mixture solution was measured after 2 h of incubation using fluorescence spectrometer under the excitation wavelength of 315 nm. The subsequent fluorescence recovery and the determination of OTA were conducted as follow: a batch of OTA with different concentration was added into the above quenched solution respectively, and the measurements of fluorescence recovery were performed after 2 h incubation. The dynamic process of fluorescence quenching and recovery was monitored by measuring the fluorescence intensity at varied incubation time. Analysis of OTA in red wine samples. Red wine samples were filtrated first to remove the wine sediment, and then were adjusted to pH 7.4 and diluted 20-fold for the subsequent analysis of OTA. Different concentrations of OTA were spiked into the red wine samples to obtain standard samples (0.05, 0.1, 0.5, 1 ng/mL). The subsequent fluorescence detection manipulation is similar to the above procedure. Characterization Methods. The morphologies of GQDs were characterized by transmission electron microscopy (TEM), performed on an instrument (FEI Tecnai G20 S-Twin) with an accelerating voltage of 200 KV. The UV-visible absorption spectra were measured on a spectrophotometer (U-3900H, Hitachi, Japan) with a variable wavelength between 200 and 400 nm. A fluorescence spectrophotometer (Hitachi F-7000) with an excitation wavelength of 315 nm and a slit width of 5 nm was used for fluorescence spectra measurement. Fluorescence lifetimes and Fluorescence quantum yields (QYs) were determined using a fluorescence spectrophotometer (FLS980, Edinburgh instruments). Infrared (IR) spectra were collected on an IR spectrometer (Nexus 670, Thermo Nicolet Corporation, American) in the wave number range of 700-4000 cm−1 using KBr pellets. The X-ray photoelectron spectroscopy (XPS) analyses were conducted using a ESCALAB 250 X-ray photoelectron spectrometer. The circular dichroism (CD) spectra were collected by a JASCO-810 spectrometer with scanning range from 230 nm to 320 nm. The HPLC-MS analysis was performed on a Shimadzu LC-20A system equipped with AB Sciex QTRAP 5500.



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RESULTS AND DISCUSSION Characterizations of GQDs and GQDs-ssDNA. Before conducting the fluorescent detection, we first characterized our synthesized GQDs and GQDs-ssDNA (ssDNA: single-stranded DNA). Figure S1A shows the typical transmission electron microscopy image of GQDs. They have almost identical sizes with the size distribution in the range of 2-5 nm. High-resolution TEM (HRTEM) image reveals that GQDs are crystalline with a lattice distance of 0.24 nm, corresponding to the (100) plane of graphite (Inset of Figure S1A).26 XPS spectra (Figure S1C) were further used to analyze the chemical composition of GQDs, revealing GQDs are mainly composed of carbon and oxygen from the predominated C1s peak and O1s peak, with C/O atomic ratio of ca. 2.24. The C 1s spectra of GQDs (Figure S1D) could be deconvoluted into three peaks at 284.5, 286.2, and 288.5 eV, associated with CC/C=C, C-O (epoxyl and hydroxyl), and C=O (carbonyl and carboxyl) groups, respectively.27 The presence of oxygen-containing groups related with carboxyl group on GQDs provides excellent water solubility as well as reaction groups for the subsequent condensation with amino-modified DNA. Figure S1E shows the fluorescence emission spectra of GQDs at different excitation wavelengths. They exhibit slightly excitation-dependent emission shifting and emission intensity change with a maximum emission peak at 433 nm when excited at the optimal wavelength of 315 nm (Figure S1F).28 The fluorescence quantum yields of the as-prepared GQDs is measured to be 8.11% by using fluorescence spectrophotometer equipped with integrating sphere detector. Figure S1G shows the UV-vis absorption of GQDs. Two typical absorption peaks at approximately 230 nm and 270 nm were observed, attributed to the π-π* transition of the aromatic sp2 domains and n-π* transition of the C=O bond, respectively.28 Besides these two absorption peaks, a new absorption peak was also observed at approximately 315 nm,29 being coincident with the excitation wavelength of strongest fluorescence emission of the GQDs. Since the fluorescent probe construction required GQDs to be connected to ssDNA through a condensation reaction, we also measured the UV-vis absorption spectra of this GQDs-ssDNA probe (e.g. GQDs-aptamer) (the red line in Figure S1G). Besides the characteristic absorption of GQDs, an obvious absorption peak concentrated at 260 nm was also observed, which represents the characteristic absorption of ssDNA, demonstrating the successful connection of GQDs to ssDNA. Fourier transform infrared (FTIR) spectra were also used to investigate GQDs and GQDsssDNA (Figure S1H). Both GQDs and GQDs-ssDNA showed characteristic vibration, including a broad intense stretching vibration band of C-OH at 3413 cm-1, C=C stretching vibration peaks at 1604 cm-1, the skeletal vibration from the graphene domain at 1434 cm-1, and a CO-C vibration peak at 1064 cm-1.30,31 In addition, three newly-emerging vibration peaks were evident in the

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GQDs-aptamer bioconjugate, as shown by the red line in Figure S1H. The peaks at 1132cm-1, 983 cm-1 and 929cm-1, attributed to symmetric phosphate, P=O and P-O stretching vibration of ssDNA, respectively, are observed due to the successful conjugation of GQDs with ssDNA.31,32

Figure 1. (A) Fluorescence spectra of GQDs in the presence of different components: (a) bare GQDs + GQD-cDNA, (b) GQDs-aptamer, (c) GQDs-cDNA, (d) GQDs-aptamer + GQDs-cDNA, (e) GQDs-aptamer + GQDs-cDNA + OTA. TEM images of (B) GQDs-cDNA, (C) GQDs-aptamer + GQDs-cDNA and (D) GQDs-aptamer + GQDs-cDNA + OTA.

Fluorescence detection of OTA. We evaluated the feasibility of our designed strategy combining the structure-switching signaling aptamers with fluorescent GQDs for the development of fluorescent OTA detection. As shown in Figure 1A, compared to bare GQDs with fluorescence emission at 433 nm (Figure S1F), an equal amount of GQDs-aptamer or GQDs-cDNA presented nearly identical fluorescence emission intensities concentrated at 430 nm. However, if the two components were mixed, an obvious fluorescence quenching of GQDs with a 9.5 nm red-shift was observed. As expected, the addition of OTA caused a high fluorescence recovery with 92% of the initial fluorescence intensity and the fluorescence emission peak returning to 430 nm. As a comparison, there was no apparent fluorescence quenching in the mixture of GQDs-cDNA and unmodified GQDs, since no DNA hybridization occurred (the black line of Figure 1A). Note that the fluorescence of the unmodified GQDs was, however, scarcely influenced by the addition of OTA (Figure S2).In order to further verify the fluorescence transformation mechanism, we used the TEM techniques to investigate the aggregation state of different GQDs, including single GQDs-cDNA, and the mixture of GQDs-aptamer and GQDs-cDNA in the absence and presence of OTA (Figure 1B-D). It is clearly obvious that the GQDs-cDNA were well-dispersed before the addition of GQDs-aptamer (Figure 1B), but a lot of large aggregates composed of small GQDs formed in the mixture of GQDs-aptamer and GQDs-cDNA (Figure 1C).


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Then, large aggregates disappeared and GQDs-ssDNA was fully redispersed in the mixture solution after the addition of OTA into the mixture of GQDs-aptamer and GQDs-cDNA (Figure 1D). These results demonstrate that the GQDs-ssDNA aggregation state changes, adjusted by aptamer structure switching, led to off-on fluorescence switching. To further verify the sensing process based on the structure-switching of aptamer, circular dichroism (CD) spectroscopy was used to characterize the aptamer structure-switching from single strand to aptamer/cDNA duplex to aptamer/OTA complex, as shown in Figure S3 of supporting information. Firstly, we confirmed that there is not any CD signal from target OTA molecules. The CD spectrum of aptamer strand shows a positive peak at 275 nm, and a negative peak at 240nm, which indicate the typical parallel G-quadruplex structure of this OTA aptamer strand.33,34 After the addition of cDNA, the duplex composed of part of aptamer strand and cDNA presents a positive peak at 280 nm with enhanced amplitude and a negative peak at 245 nm, corresponding to the base-stacking of duplex.34-36 Up on the addition of OTA, the aptamer-OTA complex gives a distinct change in CD signal with a positive band at 290 nm corresponding to the antiparallel G-quadruplex structure and a negative peak at 250 nm, accompanied by the enhancement of amplitudes. The CD spectra results demonstrate the configuration switching of the aptamer strand in our designed strategy, which can be thus used to tuning the aggregation and disaggregation behavior of GQDs for fluorescence detection.

Figure 2. (A) UV-vis absorption spectra of (a) GQDs-cDNA and (b) GQDs-cDNA + GQDs-aptamer aggregates. (B) Fluorescence intensity decay curves of (a) monodisperse GQDs-ssDNA, (b) GQDs aggregates and (c) the re-dispersed GQDs-ssDNA in the presence of OTA (20 ng/mL) as a function of time.

Fluorescence quenching mechanism. To understand the mechanism of fluorescent self-quenching of GQDs upon aggregation, we examined the UV-vis absorption and fluorescence lifetimes of dispersed GQDs-ssDNA and GQD aggregates. As mentioned above, the fluorescence emission quenching was accompanied by a 9.5 nm redshift after DNA hybridization induced GQD aggregation, which meant an energy difference between the dispersed GQDs-ssDNA and GQDs-aptamer/GQDs-cDNA aggregates. However, there was no obvious absorbance difference between the dispersed GQDs and the GQD

aggregates (Figure 2A). These data indicate a possible exciton energy transfer mechanism might play an important role, in which excition energy of higher bandgap GQDs is transferred to smaller bandgap GQDs or defect states, causing apparent fluorescent quenching and red-shift of the emission peak without absorbance changes.37,38 We also measured the fluorescence emission lifetime of the dispersed GQDs-ssDNA and GQD aggregates by monitoring the fluorescence emission intensity decay as a function of time (Figure 2B). The fluorescence intensity of these samples all followed triexponential decay kinetics. The exciton lifetime of the dispersed GQDs-ssDNA measured 7.28 ns, higher than that of the GQD aggregates (4.89 ns) upon aggregation (Table S1). Compared with the freely dispersed GQDsssDNA, the reduction of approximately 2.39 ns in the exciton lifetime of GQD aggregates could be ascribed to efficient exciton energy transfer from smaller GQDs (bigger bandgap) to larger ones (smaller bandgap) due to the shortened inter-GQD distance.39 This would result in more rapid fluorescent emission decay in the initial 20 ns, leading to fluorescence quenching and band shift. Subsequently, the exciton lifetime of the re-dispersed GQDs-ssDNA after OTA-triggered disaggregation recovered to 7.12 ns, similar to that of the original GQDsssDNA. These fluorescence lifetime results further verified the feasibility of aptamer structure-switching induced GQD aggregation and disaggregation for developing “signal-on” fluorescent sensors.

Figure 3. (A) Fluorescence quenching of the GQDsaptamer/GQDs-cDNA mixture and (B) fluorescence recovery of GQDs aggregates triggered by 20 ng/mL OTA as a function of incubation time.

High-sensitivity aptasensors for OTA detection. The fluorescence sensing process for OTA detection involved two steps: 1) aptamer-cDNA hybridization induced GQD aggregation (fluorescence quenching) and, 2) OTA triggered disaggregation of GQD aggregates (fluorescence recovery). To optimize the fluorescence detection conditions, the kinetic processes of fluorescence quenching and recovery were studied by measuring the time-dependent fluorescence emission spectra. Figure 3A shows the fluorescence quenching of the GQDs-aptamer/GQDs-cDNA mixture as a function of incubation time. The black line exhibits a rapid decline in the first 30 min followed by a slow decrease of 20 min and reached to the quenching equilibrium after 50 min. The subsequent fluorescence recovery was also a time-



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dependent process due to the preferential formation of the aptamer-OTA complex over aptamer-cDNA (Figure 3B). The best fluorescence recovery was obtained after 40 min incubation. Hence, the optimum incubation times for fluorescence quenching and recovery were 50 min and 40 min respectively. We investigated the performances of this biosensor by monitoring the fluorescence emission spectrum after introducing different concentrations of OTA. As shown in Figure 4A, the fluorescence emission intensity enhanced dramatically with the addition of OTA from 0 to 20 ng/mL, accompanied with an obvious blue shift of maximum emission wavelength to the initial wavelength position of individual GQDs-ssDNA. A curve of fluorescence intensity as a function of OTA concentration was plotted in Figure 4B. It showed that the enhancement of fluorescence intensity reached a saturation point, approaching the initial fluorescence intensity of GQDs-ssDNA, which suggests that almost all the GQDs aggregates had been transformed into individual GQDs after disaggregation triggered by OTA at the concentration of more than 20 ng/mL. The inset of Figure 4B shows that the fluorescence intensity was linear with the concentration of OTA in the range of 0-1 ng/mL. The fitting equation obtained from the linear curve can be expressed as y=73.55+114.3COTA with R2=0.9968 (COTA: OTA concentration). The detection limit (LOD) based on signal to noise ratio of 3 was calculated to be 13 pg/mL, comparable to or better than those of the most reported detection methods for OTA listed in Table 1.

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including OTA. These results indicate that this aptasensor has a high selectivity for target OTA, which can be attributed to the specific recognition and strong combining capability of aptamer toward target OTA.

Figure 5. Selectivity of the designed sensing system for OTA. The concentrations of OTA and other interferes were 20 ng/mL.

We further investigated the feasibility and reliability of this sensing platform for OTA determination in red wine samples by first spiking the samples with known concentrations of OTA. As shown in Table S2, our detection results for OTA in the red wine samples were consistent with the theoretical addition values and the results obtained from HPLC-MS technique, with 94.4102.7% of recoveries and relative standard deviation (RSD) in the range of 2.9-5.8%. The experimental results verified that the proposed sensing system can be applied to the measurement of OTA in real food samples with favorable reliability. Table 1. Comparison with the currently reported methods for OTA determination.

Figure 4. (A) The fluorescence recovery of GQDs aggregates after incubation with various concentrations of OTA (0-20 ng/mL). (B) The relationship between the fluorescence intensity and the concentration of OTA. The inset shows a 2 linear relationship (R =0.9968) with the concentration of OTA in the range of 0-1 ng/mL. Error bars were obtained from three parallel experiments.

To evaluate the selectivity of this sensing system for OTA, we compared the fluorescence response of this aptasensor toward OTA and other interferes (OTB, AFB1 and ZEN) at the same concentration (20 ng/mL; Figure 5). Only OTA could produce fluorescence recovery, while all other toxins presented negligible effects on the fluorescence signal of GQD aggregates compared with the blank control. Besides, the fluorescence recovery signals arising from OTA are not interfered by the presence of other mycotoxins in the mixture of different mycotoxins

Biosensing

Detection range

LOD of OTA

Ref.

principle Electrochemical

0.1-20 ng/mL

30 pg/mL

40

0.15-5 ng/mL

0.07 ng/mL

41

ELISA

125-8000 pg/mL

103.2 pg/mL

42

Colorimetric

0.5-100 ng/mL

30 pg/mL

43

0.5-20 ng/mL

0.22 ng/mL

44

0.05-100 ng/mL

0.02 ng/mL

45

0-20 ng/mL

1.9 ng/mL

46

0.1-1 ng/mL

20 pg/mL

47

0.05-20 ng/mL

13 pg/mL

This work

Fluorescent

CONCLUSION A fluorescence switch-on aptasensor was designed for high-sensitivity detectionof OTA based on the strategy of combining a structure-switching signaling aptamer with


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GQD aggregation/disaggregation properties. GQDs were applied as a fluorescent tag, and formation of the primary aptamer-cDNA duplex enabled the aggregation of GQDs, leading to fluorescence quenching. The introduction of target OTA then triggered the aptamer structure to switch from an aptamer-cDNA duplex to an aptamerOTA complex, resulting in subsequent disaggregation of GQDs and the corresponding fluorescence recovery. This target-triggered fluorescence signal-on strategy provides a feasible approach to quantitative detection of OTA with high sensitivity, selectivity and a detection limit of 13 pg/mL, as well as satisfactory recoveries ranging from 94.4-102.7%in practical red wine samples. This design strategy can be used not only for OTA detection, but also can be further expanded to other detection systems based on the design of structure-switching aptamers specific to the target molecule, which may open a new avenue in the construction of GQD-based aptasensors with high sensitivity and the characteristic advantages of a signal switch-on system.

ASSOCIATED CONTENT CONTENT The Supporting Information is available free of charge on the ACS Publications website. The corresponding characterizations of GQDs and GQDsDNA conjugates; Fluorescence spectra of unmodified GQDs before and after the addition of OTA; circular dichroism (CD) spectra of aptamer strand in different structures; Fluorescence lifetime fitting parameters for different samples (Table S1); Detection of OTA in red wine samples (Table S2).

AUTHOR INFORMATION Corresponding Author *Email: [email protected] *Email: [email protected]

Author Contributions The authors declare no competing financial interest.

Acknowledgements This work was financially supported by the National Natural Science Foundation of China (51672022, 51302010 and 51671003), the Specialized Research Fund for the Doctoral Program of Higher Education (20130010120009), the start-up fundings from Peking University and Young Thousand Talented Program.

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9

Disaggregation Behavior of Graphene

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