Dual-Target Electrochemical Biosensing Based on DNA Structural

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Dual-target electrochemical biosensing based on DNA structural switching on gold nanoparticle-decorated MoS2 nanosheets Shao Su, Haofan Sun, Wenfang Cao, Jie Chao, Hongzhen Peng, Xiaolei Zuo, Lihui Yuwen, Chunhai Fan, and Lianhui Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b12833 • Publication Date (Web): 03 Mar 2016 Downloaded from http://pubs.acs.org on March 4, 2016

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ACS Applied Materials & Interfaces

Dual-target electrochemical biosensing based on DNA structural switching on gold nanoparticle-decorated MoS2 nanosheets Shao Su,*, † Haofan Sun, † Wenfang Cao, † Jie Chao, † Hongzhen Peng, ‡ Xiaolei Zuo, ‡

†

Lihui Yuwen, † Chunhai Fan†, ‡ and Lianhui Wang*, †

Key Laboratory for Organic Electronics and Information Displays & Institute of

Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, 9 Wenyuan Road, Nanjing 210023, China. ‡

Division of Physical Biology, Shanghai Institute of Applied Physics, Chinese

Academy of Sciences, Shanghai 201800, China.

ABSTRACT: A MoS2-based electrochemical aptasensor has been developed for the simultaneous detection of thrombin and adenosine triphosphate (ATP) based on gold nanoparticles-decorated MoS2 (AuNPs-MoS2) nanocomposites. Two different aptamer probes labeled with redox tags were simultaneously immobilized on an AuNPs-MoS2 film modified electrode via Au-S bonds. The aptamers presented structural switches with the addition of target molecules (thrombin and ATP), resulting in methylene blue (MB) far from or ferrocene (Fc) close to the electrode surface. Therefore, a dual signaling detection strategy was developed, which featured both “signal-on” and “signal-off” elements in the detection system because of the target-induced structure switching. This proposed aptasensor could simultaneously determine ATP and thrombin as low as 0.74 nM ATP and 0.0012 nM thrombin with high selectivity,

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respectively. In addition, thrombin and ATP could act as inputs to activate an AND logic gate. KEYWORDS: Aptasensor; Molybdenum disulfide; Gold nanoparticles; Simultaneous detection; Logic gates. Introduction An aptamer is an artificial single-stranded DNA or RNA oligonucleotide, which has been employed extensively in electrochemical sensors due to its unique advantages, such as high specificity and efficient binding affinity with the targets, higher stability than antibodies and ease of chemical decoration.1-3 Usually, the structure or conformation of the aptamer is changed when a target molecule is recognized, which results in redox tags (e.g., ferrocene (Fc) or methylene blue (MB)) that modify on the aptamers close to or far from the electrode surface.4 Therefore, two sensing mechanisms (“signal-on” and “signal-off”) are often involved in constructing aptamer-based electrochemical sensors for individual or simultaneous detection of target molecules ranging from small molecules (e.g. cocaine or adenosine),5-6 proteins (e.g. thrombin or lysozyme)7-8 and metal ions (e.g. K+ or Hg2+).9-10 A “signal-on” or “signal-off’ sensing mechanism is selected to determine various targets for different purposes because of their unique advantages, such as a reagentless process, facile manipulation, few false negative results for “signal-off” sensors11 and a low background current, a high signal intensity without theoretical limits for “signal-on” sensors.12 Unfortunately, most of aptasensors only use the “signal-on” or “signal-off” mode for multiplex detection.13-14 The use of only one signal change mode easily


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causes misreading in the multiplex detection system because of the false negative or positive results. Therefore, the use of two sensing mechanisms in a detection system has attracted the attention of researchers. Recently, Grabowska et al. developed a dual genesensor for the simultaneous detection of two main markers (hemagglutinin and neuraminidase) of the influenza virus based on two types of analytical signals according to the “signal-on” and “signal-off” modes. This duo-sensor efficiently reduced misreading because it is difficult for double mutants to simultaneously appear in two restricted regions of separated targets.15 Xia and co-workers designed a 1-to-2 decoder and a 2-to-3 decoder device based on a DNA-biomolecular “signal-on” and “signal-off” sensing mechanism. These devices worked well if the corresponding DNA targets were added to the designated strategy, suggesting that the decoder was practical and easily be used as computing device.16 To obtain a better performance, nanomaterials, such as noble metal nanomaterials (e.g. Au, Ag and Pt nanoparticles)17-19, carbon nanotubes20, quantum dots21 and graphene22-23, have been introduced to improve the sensitivity of electrochemical sensors due to their unique electronic and catalytic properties. Molybdenum disulfide (MoS2) is a layered nanomaterial that has attracted the interest of an increasing number of scientists because of its graphene-like structure and unique properties.24 MoS2 is a semiconducting analog of graphene, which has an interesting indirect-to-direct bandgap transition (from 1.2 to 1.9 eV), depending on its thickness.25-26 This phenomenon may offer a possible solution to overcome the shortages of graphene, thus increasing the potential uses of MoS2 as a material for



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applications in sensing combined with electrochemistry, fluorescence and surface-enhanced Raman scattering techniques.27-29 Similar to graphene, MoS2 possesses high fluorescence quenching efficiency. Therefore, MoS2 has been used as nanoprobes for the homogeneous detection of biomolecules.30 MoS2 is easily hybridized with graphene to form nanocomposites, which could be used as nanoprobes

to

greatly

enhance

the

electrochemical

performance

of

nanocomposites-based sensors.31 As expectedly, MoS2 is also a promising supporting material that stabilizes metallic (such as Au, Ag, Pd and Pt) nanoparticles to form hierarchical

nanocomposites.32-33

Such

metal

nanoparticles-decorated

MoS2

nanocomposites possess the intrinsic properties of pure metal nanoparticles and MoS2 nanosheet due to their synergistic effect, making the MoS2-based nanocomposites exhibit

excellent

electrochemical

properties.

For

example,

platinum

nanoparticles-decorated MoS2 nanosheet showed excellent electrocatalytic ability for methanol oxidation.33 Gold nanoparticles (AuNPs)-decorated MoS2 nanocomposites have been extensively employed in construction of electrochemical sensors for neurotransmitters,34-35 glucose,36 hydrogen peroxide,37 DNA38 and proteins39 detection because of their excellent conductivity and large surface area. Inspired by these exciting studies, a dual-target aptasensor was developed for the simultaneous detection of ATP and thrombin using both “signal-on” and “signal-off” sensing mechanisms based on MoS2-based modified electrode. As shown in Figure 1, two different aptamer probes labeled with Fc and MB (hairpin and double-strand structures) were simultaneously immobilized on the AuNPs-MoS2 nanocomposites


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via an Au-S bond. ATP and thrombin were selected as the model targets, which represented a small molecule and a protein, respectively. Upon incubation with ATP and thrombin, the structure of aptamers changed with Fc close to and MB far away from electrode surface, respectively, resulting in a “signal-on” and a “signal-off” sensing mechanism. This aptasensor possessed both of the advantages of two sensing mechanisms and produced few misreadings and high sensitivity. Moreover, a convenient electronic logic gate was also designed based on this detection strategy. This as-prepared aptasensor has a great potential to provide simple, sensitive and cost-effective detection of various aptamer specific binding targets.

Scheme 1. Schematic description of the aptasensor for determination of ATP and thrombin. Experimental Reagents Molybdenum (IV) sulfide powder (< 2 µm, 99%), gold (III) tetrachloride trihydrate (HAuCl4·3H2O, ≥99%), thrombin, adenosine triphosphate (ATP), uridine triphosphate (UTP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), l-lysine, bull serum albumin (BSA), hemoglobin (Hb) and l-histidine (l-his) were purchased from Sigma-Aldrich (USA). Thrombin binding aptamer (TBA), ATP aptamer (ATPA) and its complementary sequence were purchased from TaKaRa (Dalian, China) and the



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sequences were listed as follows: TBA: 5'-SH-(CH2)6-GGTTGGTGTGGTTGGATTGATCGTAGGTACAACC-MB-3’ ATPA: 5’-SH-(CH2)6-ACCTGGGGGAGTATTGCGGAGGAAGGT-Fc-3’ ATPA complementary sequence: 5’-ACCTTCCTCCGCAATACTCCCCCAGGT-3’ All other reagents were of analytical grade and used without further purification. All solutions were prepared with Milli-Q water. Apparatus The morphologies of the MoS2 nanosheet and the AuNPs-MoS2 nanocomposite were observed using a scanning electron microscope (SEM, Hitachi S-4800) and transmission electron microscope (TEM, Hitachi H-7500). Atomic force microscope (AFM, Bruker) was employed to examine the height of MoS2 nanosheet. The software “Nano Measurer” was used to measure the diameter of AuNPs that decorated on the surface of the MoS2 nanosheet. X-ray photoelectron spectroscopy (XPS) was recorded on a PHI 5000 Versa Probe with Al Kα as the excitation source. Preparation of MoS2 and AuNPs-MoS2/GCE MoS2 nanosheet was prepared by using the same method, which reported in our previous work.26 Briefly, we firstly obtained MoS2 nanosheets using the method of intercalation exfoliation developed by Joensen.40 MoS2 (0.3 g) was applied to intercalate with 10 mL n-butyllithium solution at room temperature for two days. After washing, deoxidized water was applied to exfoliate the Li intercalated MoS2. In order to facilitate the exfoliation process, the suspension was sonicated for 1 h. Then, the LiOH and other soluble impurities were removed from MoS2 nanosheets by


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centrifuging at least twice. Finally, the product was stored at 4ºC until further application. The electrode was cleaned as follows: a 3 mm diameter glass carbon electrode (GCE) was mechanically polished using 0.3 mm and 0.05 mm alumina powders, respectively. Then, the GCE was sonicated in ethanol and ultrapure water for 1 min, respectively. After drying under a nitrogen stream, 5 µL as-prepared MoS2 nanosheets solution was dropped onto the cleaned GCE. The exfoliated MoS2 nanosheet possesses large numbers of negative charges that could effectively prevent MoS2 nanosheet aggregation due to the electrostatic repulsion. Therefore, a MoS2 film could be formed at the surface of the GCE after drying in the ambient air for about 16 h. This electrode was defined as MoS2/GCE. Then, the MoS2/GCE was immersed in 0.5 mM HAuCl4 to obtain AuNPs-MoS2/GCE via an electrodeposition method. The electrodeposition parameters were applied 10 mV s-1 of scan rate, 60 s of deposition time and pulse potential ranging from 1.1 V to -0.2 V. Immobilization of ds-ATPA and TBA on AuNPs-MoS2/GCE The AuNPs-MoS2/GCE was incubated in 0.5 µM aptamer mixture (containing 1.0 µM TBA, 1.0 µM ds-ATPA, 10 mM Tris-HCl and 0.1 mM TCEP, pH 7.4) for 12 h at room temperature. After washing, the decorated electrode was immersed in 1.0 mM 6-mercaptohexanol for 1 h to block the uncovered spots of AuNPs-MoS2/GCE. Finally, the modified electrode was washed with ultrapure water and dried using a mild nitrogen stream. Measurement procedure



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Normal pulse voltammetry (NPV), cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV) were carried out with an Autolab PGSTAT302 (Metrohm China Ltd, Switzerland). A conventional three-electrode system was used in this work. Different modified glassy carbon electrodes (GCE) were used as the working electrodes, a platinum wire and a saturated calomel electrode (SCE) were used as the counter and reference electrode, respectively. Tris hydroxymethyl aminomethane hydrochloride (Tris-HCl) buffer (pH 7.4, 10 mM) was employed as the supporting electrolyte unless specifically indicated. Electrolyte solutions were deoxygenated with nitrogen bubbling for at least 30 min, and then the solution was kept under a nitrogen atmosphere during electrochemical measurements. EIS measurement was performed in 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) containing 0.1 M KCl. The applied potential was 170 mV versus SCE. The alternative voltage amplitude was 5 mV and the voltage frequencies were ranged from 0. 01 Hz to 100 kHz. The electrochemical signals of MB and Fc were studied by SWV in the potential ranging from -0.5 to 0.7 V under the following condition: a step potential of 5 mV, a frequency of 25 Hz, and an amplitude of 20 mV. Results and discussion Characterization of MoS2 and AuNPs-MoS2 The morphology of MoS2 nanosheet was characterized by TEM and SEM. TEM (Fig. S1) and SEM (Fig. 1A) images showed that the exfoliated MoS2 nanosheet was a typical layered nanostructure. To determine the layer, AFM was used to examine the


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thickness of the exfoliated MoS2 nanosheet. Fig. S2 showed that the average height of MoS2 nanosheet was about 2.05 nm, indicating that the exfoliated MoS2 nanosheet was dilayer.41 As shown in Fig. 1B, AuNPs with an average diameter of 10 nm (Fig. S3B) were dispersed homogeneously onto the surface of MoS2 nanosheets after an electrodeposition process, indicating AuNPs-MoS2 nanocomposite was successfully synthesized (the high-resolution SEM image of AuNPs-MoS2 nanocomposite was characterized by Hitachi SU8220, which shown in Fig. S3A). Energy-dispersive X-ray spectroscopy (EDX) also confirmed the presence of gold in the nanocomposite (Fig. S4). The chemical composition of AuNPs-MoS2 nanocomposite was investigated by XPS. As shown in Fig. S5A, binding energy peaks for Au 4f were not obtained in MoS2 nanosheet. Two binding energy peaks appeared at 83.0 eV and 86.6 eV in AuNPs-MoS2 nanocomposite, which were ascribed to Au 4f7/2 and Au 4f5/2, respectively. There was a 3.6 eV difference between the Au 4f7/2 and Au 4f5/2 peaks, which was consistent with the value of zerovalent gold in the handbook.42 The S 2p3/2 and S 2p1/2 characteristic binding energy peaks were located at 160.5 eV and 161.7 eV in AuNPs-MoS2 nanocomposite, and they presented a small blue shift compared with the S 2p in MoS2 nanosheet. This result demonstrated that AuNPs had successfully grown on the surface of MoS2, resulting in changes in the binding energy peaks of S 2p. More interestingly, the binding energy peaks of Au 4f and S 2p were agreement with the binding energy peaks of thiolates adsorbed on gold, suggesting AuNPs electrodeposited on MoS2 surface via an Au-S bond (Fig. S5B).43 Some studies also shown that AuNPs were easily decorated on the surface of MoS2 nanosheet via an



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Au-S bond.28, 44 Aptamer immobilized on the surface of AuNPs-MoS2 nanocomposite (Apt-AuNPs-MoS2) was also studied by XPS (Fig. S5). A similar XPS spectrum was obtained in Apt-AuNPs-MoS2 nanocomposite, suggesting that the Au-S bond also formed in Apt-AuNPs-MoS2 nanocomposite. Moreover, a new binding energy peak of N 1s was obtained at 399.1 eV, indicating that aptamer was successfully immobilized on the AuNPs-MoS2 nanocomposite (Fig. S5C).45

Figure 1. SEM images of the (A) MoS2 nanosheet and (B) AuNPs-MoS2 nanocomposite on ITO. Electrochemical characterization of the aptasensor CV and EIS are useful techniques for characterizing the properties of surface-modified electrode. The CV characterization of the MoS2-based aptasensor was recorded in Fig. S6. The peak currents and potentials changed with every modified step, which proved the process of the aptasensor preparation. The electron transfer resistance (Ret) was described using the semicircle diameter of EIS. As shown in Fig. 2, the bare GCE showed a very small semicircle diameter (111.10 Ω, curve a). When MoS2 was immobilized on the surface of the GCE (195.99 Ω, curve c), the semicircle diameter became bigger than that of bare GCE, indicating the electron transfer from [Fe(CN)6]3-/4- to the electrode surface was hindered due to the poor


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conductivity of MoS2 and the large numbers of negative charges on MoS2 surface.27 The semicircle diameter of AuNPs-MoS2/GCE (134.62 Ω, curve b) was smaller than that of MoS2/GCE, implying that AuNPs-decorated MoS2 possessed excellent conductivity and greatly facilitated the electron transfer. After assembling the ATPA and TBA mixture on AuNPs-MoS2/GCE, the semicircle diameter increased dramatically (373.03 Ω, curve d), which was attributed to the presence of ATPA and TBA blocked the electron transfer. When MCH was used to block the remaining active spots of ATPA&TBA/AuNPs-MoS2/GCE, the Ret was raised to 406.12 Ω (curve e). After incubating with ATP, the ds-ATPA reacted with ATP and formed a tertiary structure, which blocked the electron transfer tunnel and increased semicircle diameter (455.61 Ω, curve f). Similarly, TBA incubated with thrombin and formed a G-quadruplex at the modified electrode, resulting in semicircle diameter dramatically increased (984.48 Ω, curve g). All results proved that aptamer had been successfully immobilized on AuNPs-MoS2/GCE and the proposed detection strategy worked well.

Figure 2. Nyquist plots of (a) GCE, (b) AuNPs-MoS2/GCE, (c) MoS2/GCE, (d) ATPA&TBA/AuNPs-MoS2/GCE, (e) MCH/ATPA&TBA/AuNPs-MoS2/GCE, (f)



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MCH/ATPA&TBA/AuNPs-MoS2/GCE MCH/ATPA&TBA/AuNPs-MoS2/GCE

reacted reacted

with with

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ATP,

thrombin

in

and

(g)

5

mM

K3[Fe(CN)6]/K4[Fe(CN)6] solution containing 0.1 M KCl. Sensitivity of ATP and thrombin detection In order to achieve high sensitivity, the incubation time of aptamer probe on the AuNPs-MoS2/GCE and the reaction time between aptamer and target molecules were optimized. As shown in Fig. S7A, the maximum peak current of TBA was obtained at an the incubation time of 12 h. Similarly, the peak current of ATPA grew slowly in the range from 4 to 12 h. Because the two aptamer probe were simultaneously immobilized on a modified electrode, 12 h was chosen as the optimal incubation time. The reaction time between aptamer and target molecules was also studied. The change of peak current of the MoS2-based aptasensor increased with incubation times ranging from 0 to 60 min (Fig. S7B). If the incubation time was over 60 min, the peak current of this aptasensor reached a plateau and gradually decreased. Taking account of the efficiency, 60 min was chosen as the best reaction time. Under optimal conditions, the MoS2-based aptasensor was employed to individually and simultaneously determine ATP and thrombin. We first evaluated the individual determination performances of this aptasensor. In a ternary mixture, one species concentration changed, while the other species concentration kept constant for the selective detection at ATPA&TBA/AuNPs-MoS2/GCE by SWV. As expected, the electrochemical responses change (∆I=Itarget-Ino

) of Fc was linear with the

target

increasing ATP concentration in the range from 1 nM to 10 mM with the linear


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correlation coefficient of 0.999 (Fig. 3A). The detection limit was calculated to be 0.32 nM (Fig. 3B), which was comparable to other reports or better than some previous works.46-47 More importantly, the increased ATP concentration had almost no influence on the electrochemical signal of MB. Similarly, the peak current of MB decreased with thrombin concentration increasing (Fig. 3C). The ∆I of MB was linearly dependent on the logarithm of the thrombin concentration ranging from 0.01 nM to 10 µM with detection limit of 0.0014 nM (Fig. 3D).

Figure 3. SWV curves of the aptasensor incubated with (A) 0.1 nM-10 mM ATP (from a to j: 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 µM, 10 µM, 100 µM, 1 mM and 10 mM) and (C) 0.001 nM-100 µM thrombin (from a to j: 0 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 µM, 10 µM and 100 µM) in 10 mM Tris-HCl buffer (pH 7.4), respectively. The calibration plots of the SWV peak current change versus the logarithm of (B) ATP concentration ranging from 1 nM to 10 mM and (D) thrombin concentration ranging from 0.01 nM to 10 µM..



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The feasibility of simultaneous detection was also investigated based on the MoS2-based aptasensor. As shown in Fig. 4A, the peak current of Fc gradually increased with increasing concentration of ATP, whereas the peak current of MB was suppressed by degrees with the concentrations of thrombin increased. The ∆I of Fc increased and the ∆I of MB decreased accordingly to the logarithm of ATP and thrombin concentration in the range from 1 nM to 10 mM and 0.01 nM to 10 µM, respectively (Fig. 4B, C). The detection limit of ATP and thrombin was 0.74 nM and 0.0012 nM, respectively. A comparison of the present results with other nanomaterials-based aptasensors were listed in Table S1, suggesting that the analytical parameters including linear range and detection limit based on ATPA&TBA/AuNPs-MoS2/GCE were comparable to or better than the reported results.10, 47-49 All results confirmed that this proposed MoS2-based aptasensor could individually and simultaneously determine ATP and thrombin when they co-exist in a buffer solution.

Figure 4. (A) SWV curves of aptasensor simultaneously incubated with 0.1 nM-10 mM ATP (from a to j: 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 µM, 10 µM, 100 µM, 1 mM and 10 mM) and 0.001 nM-100 µM thrombin (from a to j: 0 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 µM, 10 µM and 100 µM) in 10 mM Tris-HCl


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buffer (pH 7.4). (B) Calibration plots of ∆I versus ATP concentration ranging from 1 nM to 10 mM and (C) Calibration plots of ∆I versus thrombin concentration ranging from 0.01 nM to 10 µM. Selectivity, reproducibility and stability of the proposed aptasensor As we know, selectivity is another critical influential factor for evaluating the performance of the proposed aptasensor. Therefore, to access the specificity of the MoS2-based aptasensor, CTP, UTP, GTP and l-lysine, BSA, Hb, l-histidine were chosen as interfering species for ATP and thrombin determination, respectively. The ∆I of the aptasensor incubation with ATP was much larger (almost 4 fold) than that of incubation with CTP, UTP and GTP under the same condition (10 nM), suggesting CTP, UTP and GTP had little effect in this experiment and the aptasensor was specific to ATP with high selectivity (Fig. 5A). A similar result was obtained that l-lysine, BSA, Hb and l-histidine have almost no influence on thrombin detection (Fig. 5B). These results implied that the MoS2-based aptasensor offered high selectivity toward ATP and thrombin. The reproducibility was examined by using five equally proposed aptasensors incubated with 100 nM ATP and thrombin under the same condition. All the electrodes displayed the similar electrochemical responses with a RSD of 5.2% and 4.8% for ATP and thrombin, respectively, indicating acceptable reproducibility of the aptasensor. Additionally, the long-term storage stability of the proposed aptasensor was also studied. The aptasensor was stored at 4 ºC in refrigerator when it not used, only a small decrease of the peak current was observed after 1 week of storage. The



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peak current of ATP and thrombin was still retained above 85% and 89% of its initial response stored at 4ºC after 27 days, respectively. These results suggested the aptasensor could be used for analytes detection with acceptable stability.

Figure 5. Selectivity of the proposed aptasensor toward (A) ATP against CTP, UTP, GTP at 10 nM, and (B) thrombin against Hb, l-lysine, BSA and l-histidine at 100 nM. Establishment of ATP and thrombin triggered AND logic gate Based on above experimental results, we designed an AND logic gate by using ATP and thrombin as inputs and electrochemical signals of Fc and MB as outputs. This logic gate was dependent on the structural conversion of the aptamer probe, which was triggered by ATP and thrombin. We defined the individual peak current enhancement of Fc or suppression of MB as “OFF” or “0” output and the simultaneous peak current enhancement of Fc and suppression of MB as “ON” or “1” output (Scheme 2). From the inset table, a “1” output was achieved only when both input were “1”. If no inputs (0, 0) or only one input (0, 1 or 1, 0), which could result in “0” output. In a word, the MoS2-based multiplexed aptasensor could also serve as an “AND” gate.


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Scheme 2. Schematic description of the MoS2-based AND logic gate for determination of ATP and thrombin. Conclusion In summary, a highly sensitive and selective aptasensor was fabricated by using an AuNPs-MoS2 modified electrode. This MoS2-based aptasensor used two different aptamer probes to recognize ATP and thrombin. Upon the addition of the respective analytes, the electrochemical signal changed with the switching of aptamer structure. The MoS2-based aptasensor could individually and simultaneously detect ATP and thrombin with a satisfactory linear range, detection limit and operational stability. Moreover, we used this aptasensor to construct an “AND” logic gate for ATP and thrombin detection. The present study demonstrated a general methodology for the development of sensitive and selective aptasensors for multiplexed analytes detection.



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ASSOCIATED CONTENT

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], [email protected]. Tel: +86 25 85866333. Notes The authors declare no competing financial interest. Acknowledgements This work was financially supported by the National Basic Research Program of China (2012CB933301), the National Natural Science Foundation of China (21305070, 21475064), the Natural Science Foundation of Jiangsu Province (BK20130861), the Sci-tech Support Plan of Jiangsu Province (BE2014719), the Program

for

Changjiang

Scholars

and

Innovative

Research

Team

in

University(IRT_15R37) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Supporting Information The additional supporting data such as TEM and AFM images of MoS2 nanosheet, HRSEM image of AuNPs-MoS2, particle distribution of AuNPs, EDX image of AuNPs@MoS2 nanocomposite, effect of the incubation time and comparison of the response characteristics of different methods for ATP and thrombin detection. This material is available free of charge via the Internet at http://pubs.acs.org.


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REFERENCES (1) Iliuk, A. B.; Hu, L.; Tao, W. A., Aptamer in Bioanalytical Applications. Anal. Chem. 2011, 83 (12), 4440-4452. (2) Jhaveri, S.; Rajendran, M.; Ellington, A. D., In Vitro Selection of Signaling Aptamers. Nat. Biotechnol. 2000, 18 (12), 1293-1297. (3) Liss, M.; Petersen, B.; Wolf, H.; Prohaska, E., An Aptamer-based Quartz Crystal Protein Biosensor. Anal. Chem. 2002, 74 (17), 4488-4495. (4) Zuo, X.; Song, S.; Zhang, J.; Pan, D.; Wang, L.; Fan, C., A Target-Responsive Electrochemical Aptamer Switch (TREAS) for Reagentless Detection of Nanomolar ATP. J. Am. Chem. Soc. 2007, 129 (5), 1042-1043. (5) Liu, H.; Xiang, Y.; Lu, Y.; Crooks, R. M., Aptamer-Based Origami Paper Analytical Device for Electrochemical Detection of Adenosine. Angew. Chem. 2012, 124 (28), 7031-7034. (6) Wen, Y.; Pei, H.; Wan, Y.; Su, Y.; Huang, Q.; Song, S.; Fan, C., DNA Nanostructure-decorated Surfaces for Enhanced Aptamer-target Binding and Electrochemical Cocaine Sensors. Anal. Chem. 2011, 83 (19), 7418-7423. (7) Yan, F.; Wang, F.; Chen, Z., Aptamer-based Electrochemical Biosensor for Label-free Voltammetric Detection of Thrombin and Adenosine. Sens. Actuators B 2011, 160 (1), 1380-1385. (8) Chen, Z.; Li, L.; Zhao, H.; Guo, L.; Mu, X., Electrochemical Impedance Spectroscopy Detection of Lysozyme Based on Electrodeposited Gold Nanoparticles. Talanta 2011, 83 (5), 1501-1506. (9) O'Sullivan,

C.

K.,

Aptamer

Conformational

Switch

as

Sensitive

Electrochemical Biosensor for Potassium Ion Recognition. Chem. Commun. 2006, (32), 3432-3434. (10) Du, Y.; Li, B.; Wei, H.; Wang, Y.; Wang, E., Multifunctional Label-free Electrochemical Biosensor Based on an Integrated Aptamer. Anal. Chem. 2008, 80 (13), 5110-5117. (11) Fan, C.; Plaxco, K. W.; Heeger, A. J., Electrochemical Interrogation of Conformational Changes as a Reagentless Method for the Sequence-Specific Detection of DNA. Proc. Natl. Acad. Sci. U. S. A. 2003, 100 (16), 9134-9137. (12) Song, S.; Wang, L.; Li, J.; Fan, C.; Zhao, J., Aptamer-Based Biosensors. TrAC Trends Anal. Chem. 2008, 27 (2), 108-117. (13) Xiang, Y.; Qian, X.; Jiang, B.; Chai, Y.; Yuan, R., An Aptamer-Based Signal-On and Multiplexed Sensing Platform for One-Spot Simultaneous Electronic Detection of Proteins and Small Molecules. Chem. Commun. 2011, 47 (16), 4733-4735. (14) Zhang, J.; Wang, L.; Zhang, H.; Boey, F.; Song, S.; Fan, C., Aptamer-Based



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

Multicolor Fluorescent Gold Nanoprobes for Multiplex Detection in Homogeneous Solution. Small 2010, 6 (2), 201-204. (15) Grabowska, I.; Malecka, K.; Stachyra, A.; Góra-Sochacka, A.; Sirko, A.; Zagórski-Ostoja, W.; Radecka, H.; Radecki, J., Single Electrode Genosensor for Simultaneous Determination of Sequences Encoding Hemagglutinin and Neuraminidase of Avian Influenza Virus type H5N1. Anal. Chem. 2013, 85 (21), 10167-10173. (16) Kang, D.; White, R. J.; Xia, F.; Zuo, X.; Vallée-Bélisle, A.; Plaxco, K. W., DNA Biomolecular-Electronic Encoder and Decoder Devices Constructed by Multiplex Biosensors. NPG Asia Mater. 2012, 4 (1), e1. (17) Kashefi-Kheyrabadi, L.; Mehrgardi, M. A., Aptamer-conjugated Silver Nanoparticles for Electrochemical Detection of Adenosine Triphosphate. Biosens. Bioelectron. 2012, 37 (1), 94-98. (18) Shahdost-fard, F.; Salimi, A.; Khezrian, S., Highly Selective and Sensitive Adenosine Aptasensor Based on Platinum Nanoparticles as Catalytical Label for Amplified Detection of Biorecognition Events Through H2O2 Reduction. Biosens. Bioelectron. 2014, 53, 355-362. (19) Wang, J.; Wang, L.; Liu, X.; Liang, Z.; Song, S.; Li, W.; Li, G.; Fan, C., A Gold Nanoparticle-Based Aptamer Target Binding Readout for ATP Assay. Adv. Mater. 2007, 19 (22), 3943-3946. (20) Zelada-Guillén, G. A.; Riu, J.; Düzgün, A.; Rius, F. X., Immediate Detection of Living Bacteria at Ultralow Concentrations Using a Carbon Nanotube Based Potentiometric Aptasensor. Angew. Chem. Int. Ed. 2009, 48 (40), 7334-7337. (21) Sharon, E.; Freeman, R.; Willner, I., CdSe/ZnS Quantum Dots-G-quadruplex/hemin Hybrids as Optical DNA Sensors and Aptasensors. Anal. Chem. 2010, 82 (17), 7073-7077. (22) Chang, H.; Tang, L.; Wang, Y.; Jiang, J.; Li, J., Graphene Fluorescence Resonance Energy Transfer Aptasensor for the Thrombin Detection. Anal. Chem. 2010, 82 (6), 2341-2346. (23) Liu, X.; Yang, Y.; Hua, X.; Feng, X.; Su, S.; Huang, Y.; Fan, Q.; Wang, L.; Huang, W., An Improved Turn-On Aptasensor for Thrombin Detection Using Split Aptamer Fragments and Graphene Oxide. Chinese J. Chem. 2015, 33 (8), 981-986. (24) Huang, X.; Zeng, Z.; Zhang, H., Metal Dichalcogenide Nanosheets: Preparation, Properties and Applications. Chem. Soc. Rev. 2013, 42 (5), 1934-1946. (25) Radisavljevic, B.; Radenovic, A.; Brivio, J.; Giacometti, V.; Kis, A., Single-Layer MoS2 Transistors. Nat. Nanotechnol. 2011, 6 (3), 147-150. (26) Eda, G.; Yamaguchi, H.; Voiry, D.; Fujita, T.; Chen, M.; Chhowalla, M.,


Page 20 of 24

Page 21 of 24

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


Photoluminescence from Chemically Exfoliated MoS2. Nano Lett. 2011, 11 (12), 5111-5116. (27) Wu, S.; Zeng, Z.; He, Q.; Wang, Z.; Wang, S. J.; Du, Y.; Yin, Z.; Sun, X.; Chen, W.; Zhang, H., Electrochemically Reduced Single-Layer MoS2 Nanosheets: Characterization, Properties, and Sensing Applications. Small 2012, 8 (14), 2264-2270. (28) Su, S.; Zhang, C.; Yuwen, L.; Chao, J.; Zuo, X.; Liu, X.; Song, C.; Fan, C.; Wang, L., Creating SERS Hot Spots on MoS2 Nanosheets with in Situ Grown Gold Nanoparticles. ACS Appl. Mater. Interfaces 2014, 6 (21), 18735-18741. (29) Balcioglu, M.; Rana, M.; Robertson, N.; Yigit, M. V., DNA-Length-Dependent Quenching of Fluorescently Labeled Iron Oxide Nanoparticles with Gold, Graphene oxide and MoS2 Nanostructures. ACS Appl. Mater. Interfaces 2014, 6 (15), 12100-12110. (30) Zhu, C.; Zeng, Z.; Li, H.; Li, F.; Fan, C.; Zhang, H., Single-Layer MoS2-Based Nanoprobes for Homogeneous Detection of Biomolecules. J. Am. Chem. Soc. 2013, 135 (16), 5998-6001. (31) Jing, P.; Yi, H.; Xue, S.; Chai, Y.; Yuan, R.; Xu, W., A Sensitive Electrochemical Aptasensor Based on Palladium Nanoparticles Decorated Graphene–Molybdenum Disulfide Flower-Like Nanocomposites Enzymatic Signal Amplification. Anal. Chim. Acta 2015, 853, 234-241.

and

(32) Huang, X.; Zeng, Z.; Bao, S.; Wang, M.; Qi, X.; Fan, Z.; Zhang, H., Solution-phase Epitaxial Growth of Noble Metal Nanostructures on Dispersible Single-layer Molybdenum Disulfide Nanosheets. Nat. Commun. 2013, 4, 1444. (33) Yuwen, L.; Xu, F.; Xue, B.; Luo, Z.; Zhang, Q.; Bao, B.; Su, S.; Weng, L.; Huang, W.; Wang, L., General Synthesis of Noble Metal (Au, Ag, Pd, Pt) Nanocrystal Modified MoS2 Nanosheets and the Enhanced Catalytic Cctivity of Pd–MoS2 for Methanol Oxidation. Nanoscale 2014, 6 (11), 5762-5769. (34) Kodiyath, R.; Malak, S. T.; Combs, Z. A.; Koenig, T.; Mahmoud, M. A.; El-Sayed, M. A.; Tsukruk, V. V., Assemblies of Silver Nanocubes for Highly Sensitive SERS Chemical Vapor Detection. J.Mater. Chem. A 2013, 1 (8), 2777-2788. (35) Sun, H.; Chao, J.; Zuo, X.; Su, S.; Liu, X.; Yuwen, L.; Fan, C.; Wang, L., Gold Nanoparticle-decorated MoS2 Nanosheets for Simultaneous Detection of Ascorbic Acid, Dopamine and Uric Acid. RSC Adv. 2014, 4 (52), 27625-27629. (36) Su, S.; Sun, H.; Xu, F.; Yuwen, L.; Fan, C.; Wang, L., Direct Electrochemistry of Glucose Oxidase and a Biosensor for Glucose Based on a Glass Carbon Electrode Modified with MoS2 Nanosheets Decorated with Gold Nanoparticles.



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

Microchim. Acta 2014, 181 (13-14), 1497-1503. (37) Chao, J.; Zou, M.; Zhang, C.; Sun, H.; Pan, D.; Pei, H.; Su, S.; Yuwen, L.; Fan, C.; Wang, L., A MoS2-Based System for Efficient Immobilization of Hemoglobin and Biosensing Applications. Nanotechnology 2015, 26 (27), 274005. (38) Cao, X., Ultra-sensitive Electrochemical DNA Biosensor Based on Signal Amplification Using Gold Nanoparticles Modified with Molybdenum Disulfide, Graphene and Horseradish Peroxidase. Microchim. Acta 2014, 181 (9-10), 1133-1141. (39) Su, S.; Zou, M.; Zhao, H.; Yuan, C.; Xu, Y.; Zhang, C.; Wang, L.; Fan, C.; Wang, L., Shape-Controlled Gold Nanoparticles Supported on MoS2 Nanosheets: Synergistic Effect of Thionine and MoS2 and Their Application for Electrochemical Label-free Immunosensing. Nanoscale 2015, 7 (45), 19129-19135. (40) Joensen, P.; Frindt, R.; Morrison, S. R., Single-layer MoS2. Mater. Res. Bull. 1986, 21 (4), 457-461. (41) Coleman, J. N.; Lotya, M.; O’Neill, A.; Bergin, S. D.; King, P. J.; Khan, U.; Young, K.; Gaucher, A.; De, S.; Smith, R. J., Two-dimensional Nanosheets Produced by Liquid Exfoliation of Layered Materials. Science 2011, 331 (6017), 568-571. (42) Moulder, J. F.; Chastain, J.; King, R. C., Handbook of X-ray Photoelectron Spectroscopy: a Reference Book of Standard Spectra for Identification and Interpretation of XPS Data. Perkin-Elmer Eden Prairie, MN: 1992. (43) Mikhlin, Y.; Likhatski, M.; Tomashevich, Y.; Romanchenko, A.; Erenburg, S.; Trubina, S., XAS and XPS Examination of the Au-S Nanostructures Produced via the Reduction of Aqueous Gold (III) by Sulfide Ions. J. Electron Spectrosc. 2010, 177 (1), 24-29. (44) Sreeprasad, T.; Nguyen, P.; Kim, N.; Berry, V., Controlled, Defect-guided, Metal-Nanoparticle Incorporation onto MoS2 via Chemical and Microwave Routes: Electrical, Thermal, and Structural Properties. Nano Lett. 2013, 13 (9), 4434-4441. (45) Hu, Y.; Zhang, L.; Zhang, Y.; Wang, B.; Wang, Y.; Fan, Q.; Huang, W.; Wang, L., Plasmonic Nanobiosensor Based on Hairpin DNA for Detection of Trace Oligonucleotides Biomarker in Cancers. ACS Appl. Mater. Interfaces 2015, 7 (4), 2459-2466. (46) He, Y.; Liao, L.; Xu, C.; Wu, R.; Li, S.; Yang, Y., Determination of ATP by Resonance Light Scattering Using a Binuclear Uranyl Complex and Aptamer Modified Gold Nanoparticles as Optical Probes. Microchim. Acta 2015, 182 (1-2), 419-426.


Page 22 of 24

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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


(47) Lin, C.; Chen, Y.; Cai, Z.; Zhu, Z.; Jiang, Y.; Yang, C. J.; Chen, X., A Label-free Fluorescence Strategy for Sensitive Detection of ATP Based on the Ligation-triggered Super-sandwich. Biosens. Bioelectron. 2015, 63, 562-565. (48) Chen, L.; Chen, Z.-N., A Multifunctional Label-free Electrochemical Impedance Biosensor for Hg2+, Adenosine Triphosphate and Thrombin. Talanta 2015, 132, 664-668. (49) Wang, K.; Liao, J.; Yang, X.; Zhao, M.; Chen, M.; Yao, W.; Tan, W.; Lan, X., A Label-free Aptasensor for Highly Sensitive Detection of ATP and Thrombin Based on Metal-enhanced PicoGreen Fluorescence. Biosens. Bioelectron. 2015, 63, 172-177.



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Dual-Target Electrochemical Biosensing Based on DNA Structural

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