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A sensitive electrochemical aptasensor based on palladium nanoparticles decorated graphene–molybdenum disulfide flower-like nanocomposites and enzymatic signal amplification
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Analytica Chimica Acta xxx (2014) xxx–xxx
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A sensitive electrochemical aptasensor based on palladium nanoparticles decorated graphene–molybdenum disulfide flower-like nanocomposites and enzymatic signal amplification Pei Jing, Huayu Yi, Shuyan Xue, Yaqin Chai, Ruo Yuan *, Wenju Xu * Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education (Southwest University), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
PDDA–G–MoS2 nanoflowers were firstly used for the fabrication of thrombin aptasensor. MoS2 was adopted to enhance the surface area of graphene and accelerate the electron transfer. GOD, PdNPs and hemin/G-quadruplex could amplify the electrochemical signal through synergetic catalysis. The proposed aptasensor displayed an improved sensitivity.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 July 2014 Received in revised form 26 September 2014 Accepted 6 October 2014 Available online xxx
In the present study, with the aggregated advantages of graphene and molybdenum disulfide (MoS2), we prepared poly(diallyldimethylammonium chloride)–graphene/molybdenum disulfide (PDDA–G–MoS2) nanocomposites with flower-like structure, large surface area and excellent conductivity. Furthermore, an advanced sandwich-type electrochemical assay for sensitive detection of thrombin (TB) was fabricated using palladium nanoparticles decorated PDDA–G–MoS2 (PdNPs/PDDA–G–MoS2) as nanocarriers, which were functionalized by hemin/G-quadruplex, glucose oxidase (GOD), and toluidine blue (Tb) as redox probes. The signal amplification strategy was achieved as follows: Firstly, the immobilized GOD could effectively catalyze the oxidation of glucose to gluconolactone, coupling with the reduction of the dissolved oxygen to H2O2. Then, both PdNPs and hemin/G-quadruplex acting as hydrogen peroxide (HRP)-mimicking enzyme could further catalyze the reduction of H2O2, resulting in significant electrochemical signal amplification. So the proposed aptasensor showed high sensitivity with a wide dynamic linear range of 0.0001 to 40 nM and a relatively low detection limit of 0.062 pM for TB determination. The strategy showed huge potential of application in protein detection and disease diagnosis. ã 2014 Published by Elsevier B.V.
Keywords: Aptasensor Graphene–molybdenum disulfide Electrochemical response Signal amplification Thrombin
1. Introduction
* Corresponding authors. Tel.: +86 23 68252277; fax: +86 23 68253172. E-mail address: [email protected] (W. Xu).
As artificially selected oligonucleotides (DNA or RNA) [1,2], aptamers can specifically bind to numerous target proteins with high affinity, and have been widely employed in sensors for ultrasensitive detection by using various analytical protocols [3–8].
http://dx.doi.org/10.1016/j.aca.2014.10.003 0003-2670/ ã 2014 Published by Elsevier B.V.
Please cite this article in press as: P. Jing, et al., A sensitive electrochemical aptasensor based on palladium nanoparticles decorated graphene– molybdenum disulfide flower-like nanocomposites and enzymatic signal amplification, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j. aca.2014.10.003
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Among these methods, electrochemical aptasensors have attracted particular attention because of the merits of fast response, high sensitivity, simple operation, miniaturization and relatively low cost [9–11]. In addition, for the purpose of further improving the sensitivity of the electrochemical aptasensors, enzyme labeling amplification strategy has been extensively adopted to realize the ultrasensitive detection of targets [12,13], owing to the fact that enzyme performs as transducer to convert biomolecular recognition event into electrochemical signal and intrinsic catalytic activity to achieve the signal amplification. Besides, graphene, as a two-dimensional (2D) nanomaterial with predominant physical and chemical properties, has attracted great attentions for bioassays [14,15]. Recently, molybdenum disulfide (MoS2) with the ability to availably induce more complicated planar electric transportation properties [16], a typical family number of transition metal dichalcogenides, possesses similar layered structure of graphene and has become a research hotspot [17–20]. Furthermore, the combination of MoS2 and other conducting carbon nanomaterials has attracted extensively attention [21,22], because the combined nanocomposites exhibit synergistic effects, such as perfect conductivity, larger surface area and excellent electrochemical performance. The synergistic effects endow these nanocomposites ability to maintain the intriguing activity of the redox probes and enzymes, which provides a general perspective in the construction of novel biosensors. Thrombin (TB), an extracellular serine protein, plays significant roles in the blood coagulation cascade, haemostasis and thrombosis [23,24]. So, it is extremely crucial to realize ultrasensitive and specific detection of TB. Herein, a sandwich-type electrochemical aptasensor for TB was developed based on palladium nanoparticles decorated poly (diallyldimethylammonium chloride)–graphene/molybdenum disulfide flower-like nanocomposites(PdNPs/PDDA–G–MoS2) and enzyme catalysis reactions. As the aggregation of graphene and MoS2, PDDA–G–MoS2 was synthesized by a facile hydrothermal method [25] and firstly acted as platform to immobilize biomolecules. Through the electrostatic attractive force, the negatively charged palladium nanoparticles (PdNPs) were decorated on the surface of the positively charged PDDA–G–MoS2. With large specific surface areas and more active sites, the prepared PdNPs/PDDA– G–MoS2 served as nanocarriers for highly dense conjugation of redox-active toluidine blue (Tb), glucose oxidase (GOD), and hemin/ G-quadruplex as HRP-mimicking DNAzyme [26] with the final formation of hemin/G-quadruplex conjugated Tb-PdNPs/PDDA–G– MoS2 (secondary aptamer), owing to the strong interaction between the amine groups (NH2) of biomolecules with PdNPs [27]. The target protein TB was sandwiched between the primary aptamer and the prepared secondary aptamer. After adding glucose, the immobilized GOD effectively catalyzed the oxidation of glucose to gluconolactone with the production of H2O2, which was further electrocatalyzed by PdNPs and hemin/G-quadruplex. Thus, the electron transport of Tb was promoted, resulting in significant enhancement of the electrochemical signal. Experiment results showed that this signal amplified strategy could effectively realize the quantitative detection of TB with a broad linear range and a relative low detection limit, which offered a promising way for simple, rapid and sensitive detection of other proteins in research and clinical applications. 2. Experimental 2.1. Reagents and material Graphene oxide (GO) was obtained from Nanjing XianFeng Nano Co. (Nanjing, China). Na2MoO42H2O and poly(diallyldimethylammonium chloride) (PDDA) were purchased from Beijing Chemical Reagent Co. (Beijing, China). Carcinoembryonic antigen (CEA)
and alpha-fetoprotein (AFP) was obtained from Biocell Company (Zhengzhou, China). Thrombin (TB), glucose oxidase (GOD), toluidine blue (Tb), hemin, palladium potassium chloride (K2PdCl4), gold chloride (HAuCl4), hemoglobin (Hb), bovine serum albumin (BSA), L-cysteine (L-cys) and human IgG were obtained from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). Glucose was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Thrombin aptamer (TBA): 50 -NH2-(CH2)6-GGTTGGTGTGGTTGG-30 was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Trishydroxymethylaminomethane hydrochloride (Tris–HCl) was provided by Roche (Switzerland). The human serum samples were obtained from the Xinqiao Hospital (Chongqing, China). Tris–HCl buffer (20 mM, pH 7.4) was prepared with 1 mM MgCl2, 1 mM CaCl2, 5 mM KCl and 140 mM NaCl, used as aptamer buffer. Phosphate buffered solution (PBS) with different pH was served as working buffer throughout the experiment, containing 0.1 M Na2HPO4, 0.1 M KH2PO4 and 0.1 M KCl. The prepared solutions were kept at 4 C before use. All of the other chemicals were of analytical grade and directly used as received. Double distilled water was used throughout this experiment. 2.2. Apparatus The electrochemical measurements were carried out with a CHI-660D electrochemical workstation (Shanghai Chenhua Instrument, China) with a three electrode system comprised of a platinum wire auxiliary, a saturated calomel electrode (SCE) reference and the modified glass carbon (GCE, F = 4 mm) working electrode. The size and morphology of materials were taken with a scanning electron microscope (SEM, S-4800, Hitachi, Tokyo, Japan). X-ray photoelectron spectroscopy (XPS) analysis was performed using the Thermo ESCALAB 250Xi spectrometer with Al Ka X-ray (1486.6 eV) as the light source (Thermoelectricity Instruments, USA). pH measurement was performed by a pH meter (MP 230, Mettler-Toledo, Switzerland). 2.3. Synthesis of PdNPs decorated PDDA–G–MoS2 flower-like nanocomposites (PdNPs/PDDA–G–MoS2) Initially, PDDA functionalized graphene–MoS2 flower-like nanocomposites (PDDA–G–MoS2) were prepared via a hydrothermal method according to the previous protocol [25]. 0.0086 g GO was ultrasonically dispersed in 10 mL double distilled water. Then, 0.075 g Na2MoO42H2O and 75 mL PDDA (20 wt%) were added into GO solution. After ultrasonication and stirring for 20 min, 0.2 g thiourea was added into the mixture and diluted with double distilled water to 20 mL under vigorously stirring for 1 h. Subsequently, the obtained solution was maintained at 200 C for 24 h in a 25 mL teflon-lined stainless-steel autoclave. After cooling at room temperature, the black product was gathered via centrifugation, washed with ethanol and double distilled water alternately for several times, and dried at 60 C in a vacuum drying oven. PdNPs were attached to PDDA–G–MoS2 by electrostatic adsorption between negatively charged PdNPs and positively charged PDDA–G–MoS2. 5 mg PDDA–G–MoS2 was dispersed in 5 mL double distilled water by sonication. Then, 1 mL K2PdCl4 (10 mM) was wisely dropped into the dispersion and vigorously stirred for 10 min. In sequence, 1 mL freshly prepared NaBH4 (0.1 M) was slowly added into the mixture and the resulting mixture stirred for 30 min. Following that, the prepared PdNPs/PDDA–G–MoS2 was collected by centrifugation and washed for several times with double distilled water. For the comparison of different loading capability, PDDAreduced graphene oxide (PDDA-rGO) nanocomposites were synthesized according to reference [28] by using hydrazine
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hydrate as reductant. Then, PdNPs were decorated on the surface of PDDA-rGO with the similar procedure via electrostatic interaction. Moreover, PdNPs were prepared by directly reducing the diluted K2PdCl4, using the similar step without adding the immobilization platform. 2.4. Synthesis of hemin/G-quadruplex conjugated Tb-PdNPs/PDDA–G– MoS2 (secondary aptamer) 50 mL Tb (3 mM) and 100 mL TBA (2.5 mM) were added into 1 mL PdNPs/PDDA–G–MoS2 solution. After stirring the resulting mixture overnight at 4 C, both Tb and TBA were attached onto PdNPs/ PDDA–G–MoS2 nanocomposites along with the conjugation of PdNPs and amine groups. Meanwhile, 0.5 mg hemin was dissolved into the above solution and incubated for 2 h, followed by centrifugation and washing. In order to block the remaining active sites on Tb and TBA labeled PdNPs/PDDA–G–MoS2 nanocomposites, 1 mg GOD with abundant thiol and amino groups was introduced into the solution and incubated for 40 min. After centrifugation and washing with water, the prepared hemin/Gquadruplex conjugated Tb-PdNPs/PDDA–G–MoS2 was resuspened in 1 mL PBS (pH 7.0) for further use. To investigate the signal amplification of the proposed protocol, hemin/G-quadruplex conjugated Tb-PdNPs, TBA-Tb-PdNPs/PDDA–G–MoS2, hemin/G-quadruplex conjugated Tb-PdNPs/PDDA-rGO were also prepared. 2.5. Assembly of the electrochemical sandwich-type aptasensor The fabrication process of the aptasensor and corresponding catalysis amplifying principle were shown in Fig. 1. Prior to the modification, a GCE was polished separately with 0.3 and 0.05 mm alumina slurries, followed by successive sonication in double distilled water and ethanol to obtain a mirror-like surface. The electrode was immersed in 1 mL HAuCl4 (1%) aqueous solution for electrochemical deposition at a potential of 0.2 V for 30 s to obtain nano-Au film modified electrode (depAu/GCE). Then, 20 mL TBA (2.5 mM) was attached onto the surface of depAu/GCE and incubated for 16 h at 4 C. Next, 20 mL BSA (1 wt%) was immediately enveloped onto the electrode for 40 min at room temperature to block the remaining active sites and eliminate non-specific binding effects. Subsequently, 20 mL of TB with varying concentrations in Tris–HCl buffer (pH 7.4) was coated onto the modified electrode and incubated for 40 min. Finally, 20 mL of prepared secondary
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aptamer was putted on the modified electrode and incubated for another 1 h to form the sandwich-type sensing system. After every step finishing, the obtained electrode was washed with double distilled water to remove physically absorbed species. 2.6. Experimental measurements In order to characterize the assembly process of the modified electrode, cyclic voltammetry (CV) measurements were carried out in 1 mL PBS (0.1 M, pH 7.4) containing 5.0 mM [Fe(CN)6]3/4 as redox probe with a potential range of 0.2 to 0.6 V (vs. SCE) and a scan rate of 100 mV s1. The differential pulse voltammetry (DPV) from 0 to 0.6 V (vs. SCE) with the modulation amplitude of 0.05 V, pulse width of 0.05 s and sample width of 0.0167 s, was performed in 1 mL PBS (0.1 M, pH 7.0) containing appropriate amount of glucose to record the electrochemical signals for quantitative detection of TB. 3. Results and discussion 3.1. Characterizations of different nanomaterials The general morphology of graphene oxide (GO), PDDA– G–MoS2 and PdNPs/PDDA–G–MoS2 nanomaterials was demonstrated by the provided SEM images in Fig. 2. As shown in Fig. 2A, a sheet of GO exhibits a typical wrinkled and folded sheet structure. After MoS2 was combined with graphene in the presence of PDDA, the flower-like structure of PDDA–G–MoS2 (Fig. 2B) was formed. The flower-like structure was consisted of some thinly wrapped layers obviously and which can greatly increase the specific area of the nanocomposites. From Fig. 2C, the dense coverage of PdNPs on the surface of PDDA–G–MoS2 could be observed, implying the successful synthesis of PdNPs/PDDA–G–MoS2. Moreover, X-ray photoelectron spectroscopy (XPS) measurement was employed to provide effective information on the chemical composition of as-prepared hemin/G-quadruplex conjugated Tb-PdNPs/PDDA– G–MoS2. Fig. 2 shows the characteristic peaks of Pd3d (D) at 335.78 eV and 341.78 eV, Mo3d (E) at 232.23 eV and 235.38 eV, S2p (F) at 167.83 eV, and N1s (G) at 401.68 eV, suggesting the successful immobilization of PdNPs on the PDDA–G–MoS2 flower-like nanocomposites. Since the existence of P and Fe in hemin/G-quadruplex structure, the peaks of Fe2p and P2p of the nanocomposites could be observed in Fig. 2H and I, respectively,
Fig. 1. Schematic diagrams of the prepared aptasensor and the signal amplification mechanism.
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Fig. 2. SEM images of GO (A), PDDA–G–MoS2 (B), PdNPs/PDDA–G–MoS2 (C). XPS spectra of (D) Pd, (E) Mo, (F) S, (G) N, (H) Fe and (I) P for the prepared hemin/G-quadruplex conjugated Tb-PdNPs/PDDA–G–MoS2.
where the Fe2p peaks appeared at 709.13 eV and 723.15 eV, P2p peaks appeared at 134.08 eV, providing the evidence for the immobilization of hemin/G-quadruplex. All the above results confirmed the successful synthesis of hemin/G-quadruplex conjugated Tb-PdNPs/PDDA–G–MoS2.
3.3. Optimization of experimental conditions for electrochemical detection To obtain a higher sensitivity and selectivity of the aptasensor, some experimental parameters, including primary TBA concentration, TB incubation time, glucose concentration and
3.2. The electrochemical characterization of the stepwise modified electrodes To gain insights on the changes in the surface features of the modified electrode during the assembly process, the CVs were recorded in [Fe(CN)6]3/4 solutions and shown in Fig. 3. As can be seen, the bare GCE exhibited a pair of well defined redox peaks (curve a). Upon the electrodeposition of HAuCl4 (1%) to the GCE, the redox current of the modified electrode apparently increased (curve b), which implies that gold nanoparticles (AuNPs) could accelerate the electron transfer. When TBA was assembled on electrode surface through Au–N affinity, the redox current decreased obviously (curve c), owing to the fact that TBA blocked the electron transfer tunnel. After the employment of BSA to block nonspecific sites, a decrease of peak current was also observed (curve d). Furthermore, after incubating with 1 nM of TB (curve e), the CV response was further decreased, owing to the inert property of protein TB.
Fig. 3. CVs of (a) bare GCE, (b) depAu/GCE, (c) TBA/depAu/GCE, (d) BSA/TBA/depAu/ GCE, (e) TB/BSA/TBA/depAu/GCE in 1 mL [Fe(CN)6]3/4 (5.0 mM, pH 7.4) at a scan rate of 100 mV s1.
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Fig. 4. The effect of different experiment parameters on the electrochemical response. (A) TBA concentration and (B) TB incubation time in 1 mL [Fe(CN)6]3/4 (5.0 M, pH 7.4), (C) glucose concentration in 1 mL PBS (0.1 M, pH 7.0), (D) pH of working solution in 1 mL PBS (0.1 M) containing 91 mM glucose.
pH of testing buffer, were optimized. 20 mL of TBA with different concentrations from 0.5 to 3.0 mM was coated onto the electrode surface and CV response of the modified electrodes were measured in 1 mL [Fe(CN)6]3/4 (5.0 mM, pH 7.4). As shown in Fig. 4A, the peak current decreased gradually with the increasing of the TBA concentration, and reached plateau regions at the concentration of 2.5 mM. So, 2.5 mM was treated as the optimum concentration of TBA. The incubation time of target TB have an important impact on the analytical performance of aptasensor. Fig. 4B displayed the
dependence of the incubation time on CV response for thrombin (1 nM) in 1 mL [Fe(CN)6]3/4 (5.0 mM, pH 7.4). The peak current decreased with the reaction time up to 40 min, and then the signal began to level off, which showed the saturation of bioaffinity between aptamer with thrombin target. Therefore, incubation time of 40 min was selected for the sandwich-type assay. The concentration of glucose plays an important role for the sensitivity and selectivity of the aptasensor. Fig. 4C illustrated the cathodic peak current (I) of the proposed aptasensor at different glucose concentrations. After incubation of 1 nM TB, the
Fig. 5. DPV responses of the different sandwich format aptasensors in the absence (a) and in the presence of 91 mM glucose (b) in 1 mL PBS (0.1 M, pH 7.0) by using different secondary aptamer: (A) hemin/G-quadruplex conjugated Tb-PdNPs, (B) TBA-Tb-PdNPs/PDDA–G–MoS2, (C) hemin/G-quadruplex conjugated Tb-PdNPs/PDDA-rGO, (D) hemin/ G-quadruplex conjugated Tb-PdNPs/PDDA–G–MoS2.
Please cite this article in press as: P. Jing, et al., A sensitive electrochemical aptasensor based on palladium nanoparticles decorated graphene– molybdenum disulfide flower-like nanocomposites and enzymatic signal amplification, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j. aca.2014.10.003
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Table 1 The performance comparison of TB aptasensors based on different methodologies. Analytical methods
Linear range/nM
Detection limit/pM
References
Colorimetric Fluorescence EIS SPR CV DPV DPV ECL DPV
0.0025–6.2 – 0.12–30 0.1–75 0.01–50 1–60 0.0073–7.3 0.005–50 0.0001–40
1.5 100 0.30 100 6.3 0.5 4.6 1.7 0.062
29 30 31 32 33 34 35 36 Our work
EIS: electrochemical impedance spectroscopy, SPR: surface plasmon resonance, CV: cyclic voltammetry, DPV: differential pulse voltammetry, ECL: electrochemiluminescent.
aptasensor was tested in 1 mL PBS (0.1 M, pH 7.0) containing glucose (1 mM) with different volume of 25, 50, 75, 100 and 125 mL. As could be seen, the peak current increased with the increasing of glucose, and then leveled off at the volume of 100 mL (equivalent to the concentration of glucose in PBS reached to 91 mM). No obvious change of the peak current could be observed after addition of 100 mL glucose. Thus, 91 mM was selected as the appropriate concentration of glucose for signal amplification. The effect of working buffer’s pH on the performance of the proposed aptasensor was also investigated in 0.1 M PBS (5.0–9.0) with 1 nM TB in the presence of 91 mM glucose. As seen in Fig. 4D, when measured in pH 7.0 PBS, the obtained DPV signal was the maximum. So, we chose pH 7.0 as the optimal pH. 3.4. Comparison of different signal amplification strategies In order to evaluate the advantage of using PDDA–G–MoS2 and hemin/G-quadruplex in the proposed aptasensor for the signal amplification, four kinds of secondary aptamer were prepared for a comparative study, including (i) hemin/G-quadruplex conjugated Tb-PdNPs, (ii) TBA-Tb-PdNPs/PDDA–G–MoS2, (iii) hemin/G-quadruplex conjugated Tb-PdNPs/PDDA-rGO, and (iv) hemin/G-quadruplex conjugated Tb-PdNPs/PDDA–G–MoS2. The detection was based on the change of DPV current (DI) before and after addition of 91 mM glucose into PBS solution. As shown in Fig. 5, after incubated with the three secondary aptamers (i) (without graphene and MoS2), (ii) (without hemin) and (iii) (without MoS2) separately, the values of DI of three electrodes were only 2.69, 2.98 and 3.42 mA, respectively. In contrast, a larger DI (5.35 mA) was observed for the electrode incubated with the proposed secondary aptamer (iv). Such results obtained might be concluded for the following reasons: (1) The introduction of MoS2 in PDDA–G–MoS2 composites greatly enhanced the specific surface area of graphene. So the obtained PDDA–G–MoS2 were very helpful to increase the loading amount of electrocatalyst including PdNPs, hemin/G-quadruplex and GOD, which could further accelerate the electron transfer between the electrode and Tb for amplification of the electrochemical signal. (2) After adding glucose into the electrochemical cell, the immobilized GOD could rapidly oxidize glucose into gluconolactone accompanying with the generation of H2O2, which was further catalyzed by PdNPs and hemin/G-quadruplex on the proposed secondary aptamer to obtain an observably enhanced electrochemical signal of Tb. 3.5. Analytical performance of the electrochemical aptasensor Under the optimized experiment conditions, the sensitivity and dynamic range of the developed electrochemical aptasensor were studied by detecting routine samples with different
Fig. 6. DPV responses for different concentrations of target TB for the proposed aptasensor in 1 mL PBS (0.1 M, pH 7.0) containing 91 mM glucose. The inset displays the linear relationship between the DPV peak current and the logarithm of the target TB concentration.
concentrations of TB. The result shown in Fig. 6 indicated the reduction peak current of the aptasensor increased with the increment of TB concentration, as expected for a sandwich mechanism. Also from the Fig. 6 (inset), it was very obvious that a linear dependence between the DPV peak currents and the logarithm of TB concentration was acquired in the range from 0.0001 to 40 nM with a correlation coefficient of 0.9935. The corresponding regression equation could be seen as: I (mA) = 1.675 lgc (nM) 13.15. The estimated limit of detection was 0.062 pM (defined as DL = 3SB/m, where SB is the standard deviation of the blank signals, m is the slope of the corresponding calibration curve), suggesting the proposed aptasensor was of high sensitivity for TB. Additionally, compared to other methodologies for the detection of TB reported in previously literatures [29–36], the results displayed in Table 1 demonstrated that the present electrochemical aptasensor showed satisfactory electrochemical response characteristics. Taken together, the above results revealed the successful achievement of the electrochemical amplification strategy for TB detection, which might be benefited from the employment of enzyme functional PdNPs/PDDA–G–MoS2 nanocomposites. On the one hand, PdNPs/PDDA–G–MoS2 nanocomposites as nanocarriers not only increased the immobilization of redox probes and enzymes with superior activity, but also facilitated the electron transfer. On the other hand, the peroxidase-mimicking enzyme activity of hemin/G-quadruplex and PdNPs displayed distinctive effects on the electrochemical catalysis of the whole system.
Fig. 7. Selectivity investigation for TB detection against possible interferents: the blank buffer, BSA (10 nM), L-cys (10 nM), IgG (10 nM), Hb (10 nM), CEA (10 nM), AFP (10 nM) and the mixture consisting of the above interferents and TB. TB 1 (1 pM), TB 2 (1 nM), Mixture 1 (1 pM TB mixed with all interferents), Mixture 2 (1 nM TB mixed with all interferents). The error bars represent the standard deviation of three measurements.
Please cite this article in press as: P. Jing, et al., A sensitive electrochemical aptasensor based on palladium nanoparticles decorated graphene– molybdenum disulfide flower-like nanocomposites and enzymatic signal amplification, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j. aca.2014.10.003
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P. Jing et al. / Analytica Chimica Acta xxx (2014) xxx–xxx Table 2 Determination of TB added in human blood serum (n = 3) with the proposed aptasensora . Samples
Added TB/nM
Found TB/nMb
Recovery/%
RSD/%
1 2 3 4 5
0.001 0.01 0.1 1.0 5.0
0.00108 0.00962 0.103 0.989 4.79
108 96.2 103 98.9 95.8
6.3 7.3 5.6 3.1 7.4
a The experimental measurements were carried out by the DPV within the range of 0.6 to 0 V in 1 mL PBS (0.1 M, pH 7.0) containing 91 mM glucose at room temperature. b The values were the average from three measurements.
3.6. Specificity, reproducibility and stability of the electrochemical aptasensor The specificity of the electrochemical sensing protocol was evaluated by challenging the aptasensor toward other potential interferents, including BSA (10 nM), IgG (10 nM), L-cys (10 nM), Hb (10 nM), CEA (10 nM), AFP (10 nM) and comparing the peak current in the presence of interfering agents with the target TB. Fig. 7 shows that higher signal could be found for the target TB than the interfering agents. When these control groups were coexisted with TB, the DPV peak current had no significant difference with that in the presence of TB only, suggesting a good specificity of the developed aptasensor for TB detection. The reproducibility of the aptasensor was investigated by using six equally proposed aptasensors to detect 1 nM TB. The relative standard deviation (RSD) of 5.3% was obtained, indicating acceptable precision and reproducibility. Furthermore, the stability of the prepared aptasensor incubated with 1 nM TB was investigated by successive long-term storage in pH 7.0 PBS at 4 C. No obvious changes were found during the first 6 days and the response changes less than 2.3% of the initial current response. After storing for 10 days, 12 days and 15 days, we observed that current DPV intensity maintained 94.6%, 93.9%, and 92.3% of the initial values, demonstrating the good shelf storage stability of the aptasensor. 3.7. Analytical application of the aptasensor To evaluate the analytical reliability and application potential, recovery experiments were analyzed by using the standard addition method. A series of samples were prepared by spiking target TB with different concentrations into the 10-folddiluted human blood serum (obtained from Xinqiao Hospital of Chongqing, China). The obtained samples were measured using the proposed aptasensor and the data were given in Table 2. The recovery was ranging from 95.8 to 108% and the range of variation of RSD was from 3.1% to 7.4%, showing potential applicability to clinical applications. 4. Conclusions In summary, a signal-amplified sandwich-type electrochemical aptasensor has been successfully fabricated for selective quantification of TB, based on PdNPs/PDDA–G–MoS2 flower-like nanocomposites and enzymatic signal amplification. The flowerlike structure of PDDA–G–MoS2 was applied to immobilize electrochemical active molecules by introducing MoS2 to enhance the specific surface area of graphene and accelerate the electron transfer. The prepared PdNPs/PDDA–G–MoS2 with plentiful active sites and good biocompatibility could load larger amounts of electron mediator Tb, GOD and hemin/G-quadruplex bioelectrocatalytic complex. Furthermore, both hemin/G-quadruplex and
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PdNPs with high electrocatalysis activities corporate electrocatalyzed the reduction of H2O2 generated from the by the oxidization of glucose through GOD, which resulted in extremely amplification of the electrochemical signal. Moreover, the fabricated electrochemical aptasensor for TB showed a relatively low detection limit, wide linear range and satisfactory selectivity and reproducibility, and it would be attractive for protein target analysis in bioanalytical and clinic application. Acknowledgements We gratefully acknowledge the Ministry of Education of China (Project 708073), NNSF of China (21075100,21275119), Natural Science Foundation of Chongqing City (CSTC-2011BA7003, CSTC-2010BB4121) and Fundamental Research Funds for the Central Universities (XDJK2014A012). References [1] I. Willner, M. Zayats, Electronic aptamer-based sensors, Angew. Chem. Int. Ed 46 (2007) 6408–6418. [2] Y.L. Yuan, X.X. Gou, R. Yuan, Y.Q. Chai, Y. Zhuo, L. Mao, X.X. Gan, Electrochemical aptasensor based on the dual-amplification of G-quadruplex horseradish peroxidase-mimicking DNAzyme and blocking reagent-horseradish peroxidase, Biosens. Bioelectron. 26 (2011) 4236–4240. [3] H.P. Huang, Y.L. Tan, J.J. Shi, G.X. Liang, J.J. Zhu, DNA aptasensor for the detection of ATP based on quantum dots electrochemiluminescence, Nanoscale 2 (2010) 606–612. [4] V. Guieua, C. Ravelet, S. Perrier, Z.Y. Zhu, S. Cayez, E. Peyrin, Aptamer enzymatic cleavage protection assay for the gold nanoparticle-based colorimetric sensing of small molecules, Anal. Chim. Acta 706 (2011) 349–353. [5] S.J. Lee, B.S. Youn, J.W. Park, J.H. Niazi, Y.S. Kim, M.B. Gu, ssDNA aptamer-based surface plasmon resonance biosensor for the detection of retinol binding protein 4 for the early diagnosis of type 2 diabetes, Anal. Chem. 80 (2008) 2867–2873. [6] S. Tombelli, M. Minunni, E. Luzi, M. Mascini, Aptamer-based biosensors for the detection of HIV-1 tat protein, Bioelectrochemistry 67 (2005) 135–141. [7] J.R. Chen, X.X. Jiao, H.Q. Luo, N.B. Li, Probe-label-free electrochemical aptasensor based on methylene blue-anchored graphene oxide amplification, J. Mater. Chem. B 1 (2013) 861–864. [8] R. Hu, W. Wen, Q.L. Wang, H.Y. Xiong, X.H. Zhang, H.S. Gu, S.F. Wang, Novel electrochemical aptamer biosensor based on an enzyme-gold nanoparticle dual label for the ultrasensitive detection of epithelial tumour marker MUC1, Biosens. Bioelectron. 53 (2014) 384–389. [9] W. Cheng, S.J. Ding, Q. Li, T.X. Yu, Y.B. Yin, H.X. Ju, G.S. Ren, A simple electrochemical aptasensor for ultrasensitive protein detection using cyclic target-induced primer extension, Biosens. Bioelectron. 36 (2012) 12–17. [10] X.L. Zuo, Y. Xiao, K.W. Plaxco, High specificity, electrochemical sandwich assays based on single aptamer sequences and suitable for the direct detection of small-molecule targets in blood and other complex matrices, J. Am. Chem. Soc. 131 (2009) 6944–6945. [11] X. Sun, F.L. Li, G.H. Shen, J.D. Huang, X.Y. Wang, Aptasensor based on the synergistic contributions of chitosan–gold nanoparticles, graphene–gold nanoparticles and multi-walled carbon nanotubescobalt phthalocyanine nanocomposites forkanamycin detection, Analyst 139 (2014) 299–308. [12] J. Zhao, Y.Y. Zhang, H.T. Li, Y.Q. Wen, X.Y. Fan, F.B. Lin, L. Tan, S.Z. Yao, Ultrasensitive electrochemical aptasensor for thrombin based on the amplification of aptamer–AuNPs–HRP conjugates, Biosens. Bioelectron. 26 (2011) 2297–2303. [13] D.W. Zhang, C.J. Sun, F.T. Zhang, L. Xu, Y.L. Zhou, X.X. Zhang, An electrochemical aptasensor based on enzyme linked aptamer assay, Biosens. Bioelectron. 31 (2012) 363–368. [14] L. Jiang, J. Qian, X.W. Yang, Y.T. Yan, Q. Liu, K. Wang, K. Wang, Amplified impedimetric aptasensor based on gold nanoparticles covalently bound graphene sheet for the picomolar detection of ochratoxin A, Anal. Chim. Acta 806 (2014) 128–135. [15] X. Sun, F.L. Li, G.H. Shen, J.D. Huang, X.Y. Wang, Aptasensor based on the synergistic contributions of chitosan–gold nanoparticles, graphene–gold nanoparticles and multi-walled carbon nanotubes cobalt phthalocyanine nanocomposites for kanamycin detection, Analyst 139 (2014) 299–308. [16] K.J. Huang, Y.J. Liu, H.B. Wang, Y.Y. Wang, Y.M. Liu, Sub-femtomolar DNA detection based on layered molybdenum disulfide/multi-walled carbon nanotube composites, Au nanoparticle and enzyme multiple signal amplification, Biosens. Bioelectron. 55 (2014) 195–202. [17] H.T. Wang, Z.Y. Lu, D.S. Kong, J. Sun, T.M. Hymel, Y. Cui, Electrochemical tuning of MoS2 for efficient hydrogen evolution, ACS Nano 8 (2014) 4940–4947. [18] X.F. Zhang, B. Luster, A. Church, C. Muratore, A.A. Voevodin, P. Kohli, S. Aouadi, S. Talapatra, Carbon nanotube-MoS2 composites as solid lubricants, ACS Appl. Mater. Interfaces 1 (2009) 735–739.
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Analytica Chimica Acta xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
A sensitive electrochemical aptasensor based on palladium nanoparticles decorated graphene–molybdenum disulfide flower-like nanocomposites and enzymatic signal amplification Pei Jing, Huayu Yi, Shuyan Xue, Yaqin Chai, Ruo Yuan *, Wenju Xu * Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education (Southwest University), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
PDDA–G–MoS2 nanoflowers were firstly used for the fabrication of thrombin aptasensor. MoS2 was adopted to enhance the surface area of graphene and accelerate the electron transfer. GOD, PdNPs and hemin/G-quadruplex could amplify the electrochemical signal through synergetic catalysis. The proposed aptasensor displayed an improved sensitivity.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 July 2014 Received in revised form 26 September 2014 Accepted 6 October 2014 Available online xxx
In the present study, with the aggregated advantages of graphene and molybdenum disulfide (MoS2), we prepared poly(diallyldimethylammonium chloride)–graphene/molybdenum disulfide (PDDA–G–MoS2) nanocomposites with flower-like structure, large surface area and excellent conductivity. Furthermore, an advanced sandwich-type electrochemical assay for sensitive detection of thrombin (TB) was fabricated using palladium nanoparticles decorated PDDA–G–MoS2 (PdNPs/PDDA–G–MoS2) as nanocarriers, which were functionalized by hemin/G-quadruplex, glucose oxidase (GOD), and toluidine blue (Tb) as redox probes. The signal amplification strategy was achieved as follows: Firstly, the immobilized GOD could effectively catalyze the oxidation of glucose to gluconolactone, coupling with the reduction of the dissolved oxygen to H2O2. Then, both PdNPs and hemin/G-quadruplex acting as hydrogen peroxide (HRP)-mimicking enzyme could further catalyze the reduction of H2O2, resulting in significant electrochemical signal amplification. So the proposed aptasensor showed high sensitivity with a wide dynamic linear range of 0.0001 to 40 nM and a relatively low detection limit of 0.062 pM for TB determination. The strategy showed huge potential of application in protein detection and disease diagnosis. ã 2014 Published by Elsevier B.V.
Keywords: Aptasensor Graphene–molybdenum disulfide Electrochemical response Signal amplification Thrombin
1. Introduction
* Corresponding authors. Tel.: +86 23 68252277; fax: +86 23 68253172. E-mail address: [email protected] (W. Xu).
As artificially selected oligonucleotides (DNA or RNA) [1,2], aptamers can specifically bind to numerous target proteins with high affinity, and have been widely employed in sensors for ultrasensitive detection by using various analytical protocols [3–8].
http://dx.doi.org/10.1016/j.aca.2014.10.003 0003-2670/ ã 2014 Published by Elsevier B.V.
Please cite this article in press as: P. Jing, et al., A sensitive electrochemical aptasensor based on palladium nanoparticles decorated graphene– molybdenum disulfide flower-like nanocomposites and enzymatic signal amplification, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j. aca.2014.10.003
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Among these methods, electrochemical aptasensors have attracted particular attention because of the merits of fast response, high sensitivity, simple operation, miniaturization and relatively low cost [9–11]. In addition, for the purpose of further improving the sensitivity of the electrochemical aptasensors, enzyme labeling amplification strategy has been extensively adopted to realize the ultrasensitive detection of targets [12,13], owing to the fact that enzyme performs as transducer to convert biomolecular recognition event into electrochemical signal and intrinsic catalytic activity to achieve the signal amplification. Besides, graphene, as a two-dimensional (2D) nanomaterial with predominant physical and chemical properties, has attracted great attentions for bioassays [14,15]. Recently, molybdenum disulfide (MoS2) with the ability to availably induce more complicated planar electric transportation properties [16], a typical family number of transition metal dichalcogenides, possesses similar layered structure of graphene and has become a research hotspot [17–20]. Furthermore, the combination of MoS2 and other conducting carbon nanomaterials has attracted extensively attention [21,22], because the combined nanocomposites exhibit synergistic effects, such as perfect conductivity, larger surface area and excellent electrochemical performance. The synergistic effects endow these nanocomposites ability to maintain the intriguing activity of the redox probes and enzymes, which provides a general perspective in the construction of novel biosensors. Thrombin (TB), an extracellular serine protein, plays significant roles in the blood coagulation cascade, haemostasis and thrombosis [23,24]. So, it is extremely crucial to realize ultrasensitive and specific detection of TB. Herein, a sandwich-type electrochemical aptasensor for TB was developed based on palladium nanoparticles decorated poly (diallyldimethylammonium chloride)–graphene/molybdenum disulfide flower-like nanocomposites(PdNPs/PDDA–G–MoS2) and enzyme catalysis reactions. As the aggregation of graphene and MoS2, PDDA–G–MoS2 was synthesized by a facile hydrothermal method [25] and firstly acted as platform to immobilize biomolecules. Through the electrostatic attractive force, the negatively charged palladium nanoparticles (PdNPs) were decorated on the surface of the positively charged PDDA–G–MoS2. With large specific surface areas and more active sites, the prepared PdNPs/PDDA– G–MoS2 served as nanocarriers for highly dense conjugation of redox-active toluidine blue (Tb), glucose oxidase (GOD), and hemin/ G-quadruplex as HRP-mimicking DNAzyme [26] with the final formation of hemin/G-quadruplex conjugated Tb-PdNPs/PDDA–G– MoS2 (secondary aptamer), owing to the strong interaction between the amine groups (NH2) of biomolecules with PdNPs [27]. The target protein TB was sandwiched between the primary aptamer and the prepared secondary aptamer. After adding glucose, the immobilized GOD effectively catalyzed the oxidation of glucose to gluconolactone with the production of H2O2, which was further electrocatalyzed by PdNPs and hemin/G-quadruplex. Thus, the electron transport of Tb was promoted, resulting in significant enhancement of the electrochemical signal. Experiment results showed that this signal amplified strategy could effectively realize the quantitative detection of TB with a broad linear range and a relative low detection limit, which offered a promising way for simple, rapid and sensitive detection of other proteins in research and clinical applications. 2. Experimental 2.1. Reagents and material Graphene oxide (GO) was obtained from Nanjing XianFeng Nano Co. (Nanjing, China). Na2MoO42H2O and poly(diallyldimethylammonium chloride) (PDDA) were purchased from Beijing Chemical Reagent Co. (Beijing, China). Carcinoembryonic antigen (CEA)
and alpha-fetoprotein (AFP) was obtained from Biocell Company (Zhengzhou, China). Thrombin (TB), glucose oxidase (GOD), toluidine blue (Tb), hemin, palladium potassium chloride (K2PdCl4), gold chloride (HAuCl4), hemoglobin (Hb), bovine serum albumin (BSA), L-cysteine (L-cys) and human IgG were obtained from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). Glucose was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Thrombin aptamer (TBA): 50 -NH2-(CH2)6-GGTTGGTGTGGTTGG-30 was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Trishydroxymethylaminomethane hydrochloride (Tris–HCl) was provided by Roche (Switzerland). The human serum samples were obtained from the Xinqiao Hospital (Chongqing, China). Tris–HCl buffer (20 mM, pH 7.4) was prepared with 1 mM MgCl2, 1 mM CaCl2, 5 mM KCl and 140 mM NaCl, used as aptamer buffer. Phosphate buffered solution (PBS) with different pH was served as working buffer throughout the experiment, containing 0.1 M Na2HPO4, 0.1 M KH2PO4 and 0.1 M KCl. The prepared solutions were kept at 4 C before use. All of the other chemicals were of analytical grade and directly used as received. Double distilled water was used throughout this experiment. 2.2. Apparatus The electrochemical measurements were carried out with a CHI-660D electrochemical workstation (Shanghai Chenhua Instrument, China) with a three electrode system comprised of a platinum wire auxiliary, a saturated calomel electrode (SCE) reference and the modified glass carbon (GCE, F = 4 mm) working electrode. The size and morphology of materials were taken with a scanning electron microscope (SEM, S-4800, Hitachi, Tokyo, Japan). X-ray photoelectron spectroscopy (XPS) analysis was performed using the Thermo ESCALAB 250Xi spectrometer with Al Ka X-ray (1486.6 eV) as the light source (Thermoelectricity Instruments, USA). pH measurement was performed by a pH meter (MP 230, Mettler-Toledo, Switzerland). 2.3. Synthesis of PdNPs decorated PDDA–G–MoS2 flower-like nanocomposites (PdNPs/PDDA–G–MoS2) Initially, PDDA functionalized graphene–MoS2 flower-like nanocomposites (PDDA–G–MoS2) were prepared via a hydrothermal method according to the previous protocol [25]. 0.0086 g GO was ultrasonically dispersed in 10 mL double distilled water. Then, 0.075 g Na2MoO42H2O and 75 mL PDDA (20 wt%) were added into GO solution. After ultrasonication and stirring for 20 min, 0.2 g thiourea was added into the mixture and diluted with double distilled water to 20 mL under vigorously stirring for 1 h. Subsequently, the obtained solution was maintained at 200 C for 24 h in a 25 mL teflon-lined stainless-steel autoclave. After cooling at room temperature, the black product was gathered via centrifugation, washed with ethanol and double distilled water alternately for several times, and dried at 60 C in a vacuum drying oven. PdNPs were attached to PDDA–G–MoS2 by electrostatic adsorption between negatively charged PdNPs and positively charged PDDA–G–MoS2. 5 mg PDDA–G–MoS2 was dispersed in 5 mL double distilled water by sonication. Then, 1 mL K2PdCl4 (10 mM) was wisely dropped into the dispersion and vigorously stirred for 10 min. In sequence, 1 mL freshly prepared NaBH4 (0.1 M) was slowly added into the mixture and the resulting mixture stirred for 30 min. Following that, the prepared PdNPs/PDDA–G–MoS2 was collected by centrifugation and washed for several times with double distilled water. For the comparison of different loading capability, PDDAreduced graphene oxide (PDDA-rGO) nanocomposites were synthesized according to reference [28] by using hydrazine
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hydrate as reductant. Then, PdNPs were decorated on the surface of PDDA-rGO with the similar procedure via electrostatic interaction. Moreover, PdNPs were prepared by directly reducing the diluted K2PdCl4, using the similar step without adding the immobilization platform. 2.4. Synthesis of hemin/G-quadruplex conjugated Tb-PdNPs/PDDA–G– MoS2 (secondary aptamer) 50 mL Tb (3 mM) and 100 mL TBA (2.5 mM) were added into 1 mL PdNPs/PDDA–G–MoS2 solution. After stirring the resulting mixture overnight at 4 C, both Tb and TBA were attached onto PdNPs/ PDDA–G–MoS2 nanocomposites along with the conjugation of PdNPs and amine groups. Meanwhile, 0.5 mg hemin was dissolved into the above solution and incubated for 2 h, followed by centrifugation and washing. In order to block the remaining active sites on Tb and TBA labeled PdNPs/PDDA–G–MoS2 nanocomposites, 1 mg GOD with abundant thiol and amino groups was introduced into the solution and incubated for 40 min. After centrifugation and washing with water, the prepared hemin/Gquadruplex conjugated Tb-PdNPs/PDDA–G–MoS2 was resuspened in 1 mL PBS (pH 7.0) for further use. To investigate the signal amplification of the proposed protocol, hemin/G-quadruplex conjugated Tb-PdNPs, TBA-Tb-PdNPs/PDDA–G–MoS2, hemin/G-quadruplex conjugated Tb-PdNPs/PDDA-rGO were also prepared. 2.5. Assembly of the electrochemical sandwich-type aptasensor The fabrication process of the aptasensor and corresponding catalysis amplifying principle were shown in Fig. 1. Prior to the modification, a GCE was polished separately with 0.3 and 0.05 mm alumina slurries, followed by successive sonication in double distilled water and ethanol to obtain a mirror-like surface. The electrode was immersed in 1 mL HAuCl4 (1%) aqueous solution for electrochemical deposition at a potential of 0.2 V for 30 s to obtain nano-Au film modified electrode (depAu/GCE). Then, 20 mL TBA (2.5 mM) was attached onto the surface of depAu/GCE and incubated for 16 h at 4 C. Next, 20 mL BSA (1 wt%) was immediately enveloped onto the electrode for 40 min at room temperature to block the remaining active sites and eliminate non-specific binding effects. Subsequently, 20 mL of TB with varying concentrations in Tris–HCl buffer (pH 7.4) was coated onto the modified electrode and incubated for 40 min. Finally, 20 mL of prepared secondary
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aptamer was putted on the modified electrode and incubated for another 1 h to form the sandwich-type sensing system. After every step finishing, the obtained electrode was washed with double distilled water to remove physically absorbed species. 2.6. Experimental measurements In order to characterize the assembly process of the modified electrode, cyclic voltammetry (CV) measurements were carried out in 1 mL PBS (0.1 M, pH 7.4) containing 5.0 mM [Fe(CN)6]3/4 as redox probe with a potential range of 0.2 to 0.6 V (vs. SCE) and a scan rate of 100 mV s1. The differential pulse voltammetry (DPV) from 0 to 0.6 V (vs. SCE) with the modulation amplitude of 0.05 V, pulse width of 0.05 s and sample width of 0.0167 s, was performed in 1 mL PBS (0.1 M, pH 7.0) containing appropriate amount of glucose to record the electrochemical signals for quantitative detection of TB. 3. Results and discussion 3.1. Characterizations of different nanomaterials The general morphology of graphene oxide (GO), PDDA– G–MoS2 and PdNPs/PDDA–G–MoS2 nanomaterials was demonstrated by the provided SEM images in Fig. 2. As shown in Fig. 2A, a sheet of GO exhibits a typical wrinkled and folded sheet structure. After MoS2 was combined with graphene in the presence of PDDA, the flower-like structure of PDDA–G–MoS2 (Fig. 2B) was formed. The flower-like structure was consisted of some thinly wrapped layers obviously and which can greatly increase the specific area of the nanocomposites. From Fig. 2C, the dense coverage of PdNPs on the surface of PDDA–G–MoS2 could be observed, implying the successful synthesis of PdNPs/PDDA–G–MoS2. Moreover, X-ray photoelectron spectroscopy (XPS) measurement was employed to provide effective information on the chemical composition of as-prepared hemin/G-quadruplex conjugated Tb-PdNPs/PDDA– G–MoS2. Fig. 2 shows the characteristic peaks of Pd3d (D) at 335.78 eV and 341.78 eV, Mo3d (E) at 232.23 eV and 235.38 eV, S2p (F) at 167.83 eV, and N1s (G) at 401.68 eV, suggesting the successful immobilization of PdNPs on the PDDA–G–MoS2 flower-like nanocomposites. Since the existence of P and Fe in hemin/G-quadruplex structure, the peaks of Fe2p and P2p of the nanocomposites could be observed in Fig. 2H and I, respectively,
Fig. 1. Schematic diagrams of the prepared aptasensor and the signal amplification mechanism.
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Fig. 2. SEM images of GO (A), PDDA–G–MoS2 (B), PdNPs/PDDA–G–MoS2 (C). XPS spectra of (D) Pd, (E) Mo, (F) S, (G) N, (H) Fe and (I) P for the prepared hemin/G-quadruplex conjugated Tb-PdNPs/PDDA–G–MoS2.
where the Fe2p peaks appeared at 709.13 eV and 723.15 eV, P2p peaks appeared at 134.08 eV, providing the evidence for the immobilization of hemin/G-quadruplex. All the above results confirmed the successful synthesis of hemin/G-quadruplex conjugated Tb-PdNPs/PDDA–G–MoS2.
3.3. Optimization of experimental conditions for electrochemical detection To obtain a higher sensitivity and selectivity of the aptasensor, some experimental parameters, including primary TBA concentration, TB incubation time, glucose concentration and
3.2. The electrochemical characterization of the stepwise modified electrodes To gain insights on the changes in the surface features of the modified electrode during the assembly process, the CVs were recorded in [Fe(CN)6]3/4 solutions and shown in Fig. 3. As can be seen, the bare GCE exhibited a pair of well defined redox peaks (curve a). Upon the electrodeposition of HAuCl4 (1%) to the GCE, the redox current of the modified electrode apparently increased (curve b), which implies that gold nanoparticles (AuNPs) could accelerate the electron transfer. When TBA was assembled on electrode surface through Au–N affinity, the redox current decreased obviously (curve c), owing to the fact that TBA blocked the electron transfer tunnel. After the employment of BSA to block nonspecific sites, a decrease of peak current was also observed (curve d). Furthermore, after incubating with 1 nM of TB (curve e), the CV response was further decreased, owing to the inert property of protein TB.
Fig. 3. CVs of (a) bare GCE, (b) depAu/GCE, (c) TBA/depAu/GCE, (d) BSA/TBA/depAu/ GCE, (e) TB/BSA/TBA/depAu/GCE in 1 mL [Fe(CN)6]3/4 (5.0 mM, pH 7.4) at a scan rate of 100 mV s1.
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Fig. 4. The effect of different experiment parameters on the electrochemical response. (A) TBA concentration and (B) TB incubation time in 1 mL [Fe(CN)6]3/4 (5.0 M, pH 7.4), (C) glucose concentration in 1 mL PBS (0.1 M, pH 7.0), (D) pH of working solution in 1 mL PBS (0.1 M) containing 91 mM glucose.
pH of testing buffer, were optimized. 20 mL of TBA with different concentrations from 0.5 to 3.0 mM was coated onto the electrode surface and CV response of the modified electrodes were measured in 1 mL [Fe(CN)6]3/4 (5.0 mM, pH 7.4). As shown in Fig. 4A, the peak current decreased gradually with the increasing of the TBA concentration, and reached plateau regions at the concentration of 2.5 mM. So, 2.5 mM was treated as the optimum concentration of TBA. The incubation time of target TB have an important impact on the analytical performance of aptasensor. Fig. 4B displayed the
dependence of the incubation time on CV response for thrombin (1 nM) in 1 mL [Fe(CN)6]3/4 (5.0 mM, pH 7.4). The peak current decreased with the reaction time up to 40 min, and then the signal began to level off, which showed the saturation of bioaffinity between aptamer with thrombin target. Therefore, incubation time of 40 min was selected for the sandwich-type assay. The concentration of glucose plays an important role for the sensitivity and selectivity of the aptasensor. Fig. 4C illustrated the cathodic peak current (I) of the proposed aptasensor at different glucose concentrations. After incubation of 1 nM TB, the
Fig. 5. DPV responses of the different sandwich format aptasensors in the absence (a) and in the presence of 91 mM glucose (b) in 1 mL PBS (0.1 M, pH 7.0) by using different secondary aptamer: (A) hemin/G-quadruplex conjugated Tb-PdNPs, (B) TBA-Tb-PdNPs/PDDA–G–MoS2, (C) hemin/G-quadruplex conjugated Tb-PdNPs/PDDA-rGO, (D) hemin/ G-quadruplex conjugated Tb-PdNPs/PDDA–G–MoS2.
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Table 1 The performance comparison of TB aptasensors based on different methodologies. Analytical methods
Linear range/nM
Detection limit/pM
References
Colorimetric Fluorescence EIS SPR CV DPV DPV ECL DPV
0.0025–6.2 – 0.12–30 0.1–75 0.01–50 1–60 0.0073–7.3 0.005–50 0.0001–40
1.5 100 0.30 100 6.3 0.5 4.6 1.7 0.062
29 30 31 32 33 34 35 36 Our work
EIS: electrochemical impedance spectroscopy, SPR: surface plasmon resonance, CV: cyclic voltammetry, DPV: differential pulse voltammetry, ECL: electrochemiluminescent.
aptasensor was tested in 1 mL PBS (0.1 M, pH 7.0) containing glucose (1 mM) with different volume of 25, 50, 75, 100 and 125 mL. As could be seen, the peak current increased with the increasing of glucose, and then leveled off at the volume of 100 mL (equivalent to the concentration of glucose in PBS reached to 91 mM). No obvious change of the peak current could be observed after addition of 100 mL glucose. Thus, 91 mM was selected as the appropriate concentration of glucose for signal amplification. The effect of working buffer’s pH on the performance of the proposed aptasensor was also investigated in 0.1 M PBS (5.0–9.0) with 1 nM TB in the presence of 91 mM glucose. As seen in Fig. 4D, when measured in pH 7.0 PBS, the obtained DPV signal was the maximum. So, we chose pH 7.0 as the optimal pH. 3.4. Comparison of different signal amplification strategies In order to evaluate the advantage of using PDDA–G–MoS2 and hemin/G-quadruplex in the proposed aptasensor for the signal amplification, four kinds of secondary aptamer were prepared for a comparative study, including (i) hemin/G-quadruplex conjugated Tb-PdNPs, (ii) TBA-Tb-PdNPs/PDDA–G–MoS2, (iii) hemin/G-quadruplex conjugated Tb-PdNPs/PDDA-rGO, and (iv) hemin/G-quadruplex conjugated Tb-PdNPs/PDDA–G–MoS2. The detection was based on the change of DPV current (DI) before and after addition of 91 mM glucose into PBS solution. As shown in Fig. 5, after incubated with the three secondary aptamers (i) (without graphene and MoS2), (ii) (without hemin) and (iii) (without MoS2) separately, the values of DI of three electrodes were only 2.69, 2.98 and 3.42 mA, respectively. In contrast, a larger DI (5.35 mA) was observed for the electrode incubated with the proposed secondary aptamer (iv). Such results obtained might be concluded for the following reasons: (1) The introduction of MoS2 in PDDA–G–MoS2 composites greatly enhanced the specific surface area of graphene. So the obtained PDDA–G–MoS2 were very helpful to increase the loading amount of electrocatalyst including PdNPs, hemin/G-quadruplex and GOD, which could further accelerate the electron transfer between the electrode and Tb for amplification of the electrochemical signal. (2) After adding glucose into the electrochemical cell, the immobilized GOD could rapidly oxidize glucose into gluconolactone accompanying with the generation of H2O2, which was further catalyzed by PdNPs and hemin/G-quadruplex on the proposed secondary aptamer to obtain an observably enhanced electrochemical signal of Tb. 3.5. Analytical performance of the electrochemical aptasensor Under the optimized experiment conditions, the sensitivity and dynamic range of the developed electrochemical aptasensor were studied by detecting routine samples with different
Fig. 6. DPV responses for different concentrations of target TB for the proposed aptasensor in 1 mL PBS (0.1 M, pH 7.0) containing 91 mM glucose. The inset displays the linear relationship between the DPV peak current and the logarithm of the target TB concentration.
concentrations of TB. The result shown in Fig. 6 indicated the reduction peak current of the aptasensor increased with the increment of TB concentration, as expected for a sandwich mechanism. Also from the Fig. 6 (inset), it was very obvious that a linear dependence between the DPV peak currents and the logarithm of TB concentration was acquired in the range from 0.0001 to 40 nM with a correlation coefficient of 0.9935. The corresponding regression equation could be seen as: I (mA) = 1.675 lgc (nM) 13.15. The estimated limit of detection was 0.062 pM (defined as DL = 3SB/m, where SB is the standard deviation of the blank signals, m is the slope of the corresponding calibration curve), suggesting the proposed aptasensor was of high sensitivity for TB. Additionally, compared to other methodologies for the detection of TB reported in previously literatures [29–36], the results displayed in Table 1 demonstrated that the present electrochemical aptasensor showed satisfactory electrochemical response characteristics. Taken together, the above results revealed the successful achievement of the electrochemical amplification strategy for TB detection, which might be benefited from the employment of enzyme functional PdNPs/PDDA–G–MoS2 nanocomposites. On the one hand, PdNPs/PDDA–G–MoS2 nanocomposites as nanocarriers not only increased the immobilization of redox probes and enzymes with superior activity, but also facilitated the electron transfer. On the other hand, the peroxidase-mimicking enzyme activity of hemin/G-quadruplex and PdNPs displayed distinctive effects on the electrochemical catalysis of the whole system.
Fig. 7. Selectivity investigation for TB detection against possible interferents: the blank buffer, BSA (10 nM), L-cys (10 nM), IgG (10 nM), Hb (10 nM), CEA (10 nM), AFP (10 nM) and the mixture consisting of the above interferents and TB. TB 1 (1 pM), TB 2 (1 nM), Mixture 1 (1 pM TB mixed with all interferents), Mixture 2 (1 nM TB mixed with all interferents). The error bars represent the standard deviation of three measurements.
Please cite this article in press as: P. Jing, et al., A sensitive electrochemical aptasensor based on palladium nanoparticles decorated graphene– molybdenum disulfide flower-like nanocomposites and enzymatic signal amplification, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j. aca.2014.10.003
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P. Jing et al. / Analytica Chimica Acta xxx (2014) xxx–xxx Table 2 Determination of TB added in human blood serum (n = 3) with the proposed aptasensora . Samples
Added TB/nM
Found TB/nMb
Recovery/%
RSD/%
1 2 3 4 5
0.001 0.01 0.1 1.0 5.0
0.00108 0.00962 0.103 0.989 4.79
108 96.2 103 98.9 95.8
6.3 7.3 5.6 3.1 7.4
a The experimental measurements were carried out by the DPV within the range of 0.6 to 0 V in 1 mL PBS (0.1 M, pH 7.0) containing 91 mM glucose at room temperature. b The values were the average from three measurements.
3.6. Specificity, reproducibility and stability of the electrochemical aptasensor The specificity of the electrochemical sensing protocol was evaluated by challenging the aptasensor toward other potential interferents, including BSA (10 nM), IgG (10 nM), L-cys (10 nM), Hb (10 nM), CEA (10 nM), AFP (10 nM) and comparing the peak current in the presence of interfering agents with the target TB. Fig. 7 shows that higher signal could be found for the target TB than the interfering agents. When these control groups were coexisted with TB, the DPV peak current had no significant difference with that in the presence of TB only, suggesting a good specificity of the developed aptasensor for TB detection. The reproducibility of the aptasensor was investigated by using six equally proposed aptasensors to detect 1 nM TB. The relative standard deviation (RSD) of 5.3% was obtained, indicating acceptable precision and reproducibility. Furthermore, the stability of the prepared aptasensor incubated with 1 nM TB was investigated by successive long-term storage in pH 7.0 PBS at 4 C. No obvious changes were found during the first 6 days and the response changes less than 2.3% of the initial current response. After storing for 10 days, 12 days and 15 days, we observed that current DPV intensity maintained 94.6%, 93.9%, and 92.3% of the initial values, demonstrating the good shelf storage stability of the aptasensor. 3.7. Analytical application of the aptasensor To evaluate the analytical reliability and application potential, recovery experiments were analyzed by using the standard addition method. A series of samples were prepared by spiking target TB with different concentrations into the 10-folddiluted human blood serum (obtained from Xinqiao Hospital of Chongqing, China). The obtained samples were measured using the proposed aptasensor and the data were given in Table 2. The recovery was ranging from 95.8 to 108% and the range of variation of RSD was from 3.1% to 7.4%, showing potential applicability to clinical applications. 4. Conclusions In summary, a signal-amplified sandwich-type electrochemical aptasensor has been successfully fabricated for selective quantification of TB, based on PdNPs/PDDA–G–MoS2 flower-like nanocomposites and enzymatic signal amplification. The flowerlike structure of PDDA–G–MoS2 was applied to immobilize electrochemical active molecules by introducing MoS2 to enhance the specific surface area of graphene and accelerate the electron transfer. The prepared PdNPs/PDDA–G–MoS2 with plentiful active sites and good biocompatibility could load larger amounts of electron mediator Tb, GOD and hemin/G-quadruplex bioelectrocatalytic complex. Furthermore, both hemin/G-quadruplex and
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Please cite this article in press as: P. Jing, et al., A sensitive electrochemical aptasensor based on palladium nanoparticles decorated graphene– molybdenum disulfide flower-like nanocomposites and enzymatic signal amplification, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j. aca.2014.10.003