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A novel label-free electrochemiluminescence aptasensor based on layered flowerlike molybdenum sulfide–graphene nanocomposites as matrix
Colloids and Surfaces B: Biointerfaces 122 (2014) 287–293
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
A novel label-free electrochemiluminescence aptasensor based on layered flowerlike molybdenum sulfide–graphene nanocomposites as matrix Yan-Ming Liu a,∗ , Gui-Fang Shi a , Jing-Jing Zhang a , Min Zhou a , Jun-Tao Cao a , Ke-Jing Huang a , Shu-Wei Ren b a b
College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000, China Xinyang Central Hospital, Xinyang 464000, China
a r t i c l e
i n f o
Article history: Received 24 March 2014 Received in revised form 2 July 2014 Accepted 8 July 2014 Available online 16 July 2014 Keywords: Molybdenum sulfide–graphene nanocomposites Electrochemiluminescence Thrombin Aptasensor
a b s t r a c t A label-free and ultrasensitive electrochemiluminescence (ECL) aptasensor was constructed for the detection of thrombin. Molybdenum sulfide–graphene nanocomposites with good conductivity and large surface area were immobilized on glassy carbon electrode (GCE), and then Nafion was fixed to chemosorb the Ru(bpy)3 2+ used as luminescence agent. Subsequently, gold nanoparticles (AuNPs) were modified on the electrode to immobilize the thiol-modified thrombin aptamer for fabrication of the thrombin aptasensor. The proposed ECL aptasensor produced the ultrasensitive detection of thrombin with a low detection limit of 3.6 × 10−15 M (S/N = 3) and over a wide target concentration range from 1.0 × 10−14 to 5.0 × 10−9 M. The aptasensor has been successfully applied in the determination of thrombin in human plasma samples of both traumatic and non-traumatic injury patients, indicating its promise in biochemical analysis. The recoveries of thrombin in human plasma samples are between 88.6% and 105.0%, and the RSD values are no more than 3.7%. The results demonstrate that this aptasensor has excellent sensitivity, selectivity and stability. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Thrombin is a kind of extracellular serine protease and involved in a number of physiological and pathological processes, such as blood coagulation, thrombosis, cardiovascular diseases, platelet activation, and even in the angiogenesis as a biomarker [1]. Thrombin of low level in organism is neuroprotective while high level is deleterious. Therefore, the determination of thrombin is very important in biochemical analysis. Aptamer as a novel capture agent has been intensively used for protein detection with the advantages of high specificity, affinity, nontoxicity, good stability, and relatively easy preparation [2,3]. Furthermore, aptamer can be easily modified with a variety of chemical groups that can provide an extraordinary flexibility in different assays and can be fixed on electrode surface through various chemical forces [4,5]. AuNPs have attracted great attentions due to their good biocompatibility, excellent conductivity and large specific surface area. The conjugates of AuNPs and thiol-modified aptamer through an Au S bond have
∗ Corresponding author. Fax: +86 376 6392889. E-mail address: [email protected] (Y.-M. Liu). http://dx.doi.org/10.1016/j.colsurfb.2014.07.011 0927-7765/© 2014 Elsevier B.V. All rights reserved.
been widely studied [6]. Up to now, various aptamer-based methods for thrombin detection have been reported, such as enhanced surface plasmon resonance [7], chronocoulometry [8], fluorescence [9], differential pulse voltammetry [10,11]. Electrochemiluminescence (ECL) has become an attractive and powerful detection tool because of its high sensitivity, good selectivity, wide linear range, and low equipment cost [12,13]. Ru(bpy)3 2+ ECL system has been successfully used in the analysis of amino acids [14], drugs [15], DNA [16] and protein [17,18] due to its excellent stability and high ECL quantum yield. The immobilization of Ru(bpy)3 2+ on electrode surface could reduce the amount of the reagent and simplify the experimental design. However, the leakage of Ru(bpy)3 2+ , resulting from its inherent water-solubility, always hinders the gain of stable ECL sensor. So far various methods for immobilization Ru(bpy)3 2+ have been developed to solve this problem [19–21]. For example, Ru(bpy)3 2+ -doped silica nanoparticles were applied to develop an aptamer-based ECL sensor for thrombin detection with a detection limit of 2.0 × 10−13 M in Lin’s work [19]. Zhang et al. [20] developed a signal-on junction-probe ECL biosensor for the detection of thrombin using AuNPs-Ru(bpy)3 2+ as signal probe. Fang et al. [21] utilized aptamer functionalized Ru(bpy)3 2+ as labels to probe the
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thrombin with the detection limit of 10 nM. In these methods, a time-consuming labeling process was involved, so novel label free Ru(bpy)3 2+ ECL biosensors are highly desired. Recently, 2-dimensional layered materials such as MoS2 , SnS2 and WS2 have been widely used as lubricants, catalysts [22], electrode materials for capacitors [23], and lithium-ion batteries [24] due to their high surface area, excellent chemical stability and strong mechanical strength. MoS2 is a family member of transition-metal dichalcogenides and is composed of Mo metal layers sandwiched between two sulfur layers [25,26]. Its layered structure is therefore expected to act as an excellent functional material because the 2-dimensional electron–electron correlations among Mo atoms would aid in enhancing planar electric transportation properties. However, few attentions have been paid on the analytical application of MoS2 as an electrode material for ECL sensor because its electrical conductivity is lower than that of carbon nanotube/graphene [27]. Graphene has excellent electrical conductivity, large surface area and chemical stability [3,28]. It has been applied in the detection of ATP with detection limit of 2.01 × 10−11 M [29]. Graphene-based nanomaterials have been extensively studied [30]. The combination of graphene and MoS2 may provide a new nanomaterial with good electrochemical properties. Our group prepared an electrochemical sensor based on MoS2 –graphene nanohybrid for acetaminophen detection with detection limit of 2.0 × 10−8 M [31]. In our previous work, we have developed a CdSe-ZnS quantum dots labeled, sandwich-type ECL aptasensor for thrombin detection [32]. CdSe-ZnS quantum dots were used as ECL emission reagent and two aptamers were used to form a sandwich-type structure. In this work, we fabricated a simple, label-free single chain aptamer aptasensor used Ru(bpy)3 2+ as the ECL reagent, and MoS2 –graphene nanocomposites with flowerlike structure as matrix to immobilize Ru(bpy)3 2+ . The thrombin was selected as model analyte. The process of the stepwise modification of GCE was investigated in detail. The high sensitive detection of thrombin in complex samples was achieved, demonstrating the great potential of the ECL aptasensor for protein detection. 2. Experimental 2.1. Reagents Ru(bpy)3 Cl2 ·6H2 O and HAuCl4 ·3H2 O were purchased from Alfa Aesar (Tianjin, China). Na2 MoO4 ·2H2 O, l-cysteine, tri-npropylamine (TPA, 98%), and citric acid trisodium salt dihydrate (C6 H5 Na3 O7 ·2H2 O) was from the Sinopharm Group Chemical Reagent Co. Ltd. (Shanghai, China). SH-aptamer (5 -SH-(CH2 )6 -TTT TTT TTT TTT TTT GGT TGG TGT GGT TGG-3 ) was synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). Human thrombin, human immunoglobulin (hIgG), human serum albumin (HSA), and bovine serum albumin (BSA, Mr = 67,000) from Shanghai Solarbio Bioscience & Technology Co. Ltd. (Seebio Biotechnology). Nafion (5 wt%) from Sigma–Aldrich (USA). Tris–HCl buffer (pH 7.4) containing 20 mM Tris, 100 mM NaCl, 5 mM KCl, 1 mM MgCl2 and 1 mM CaCl2 was used to dissolve thrombin. 0.2 M phosphate buffer saline (PBS, pH 7.4) from Sangon Biotech Co. Ltd. (Shanghai, China) was used to dissolve aptamer. All chemicals were of analytical grade. The water used was processed with an Ultrapure Water System (Kangning Water Treatment Solution Provider, China). 2.2. Apparatus Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements were carried out with RST5200
electrochemical workstation (Zhengzhou Shiruisi Technology Co. Ltd., China). A three-electrode system was composed of a modified GCE (ϕ = 3 mm) as the working electrode, a Pt spiral wire as the counter electrode and an Ag/AgCl as the reference electrode. Transmission electron microscope (TEM) images were recorded by JEM-2100F (JEOL Ltd., Tokyo, Japan) TEM system. Scanning electron microscopy (SEM) images were obtained by S-4800 (Hitachi, Tokyo, Japan). X-ray powder diffraction (XRD) pattern was operated on RigakuD/Maxr-A X-ray diffractometer (Japan). X-ray photoelectron spectroscopy (XPS) measurement was performed by Kratos Axis Ultra electron spectrometer (UK). Fourier transform infrared (FTIR) spectrum was recorded on Bruker TENZOR 27 spectrophotometer (Germany).
2.3. Preparation of MoS2 –graphene nanocomposites The graphene oxide (GO) was synthesized from graphite flake using the Hummers’ method [33]. The preparation of MoS2 –graphene nanocomposites followed the procedure of our previous work [31]. Briefly, 0.1 g GO and 50 mL water were mixed and treated with ultrasonication. Then, 0.5 g Na2 MoO4 ·2H2 O was added and ultrasonicated for 20 min and the pH value was adjusted to 6.5 with 0.1 M NaOH. Subsequently, 1.0 g l-cysteine and 40 mL water were added and then transferred into a Teflon-lined stainless steel autoclave which was heated to 180 ◦ C for 48 h. After cooling naturally, the black precipitates was collected by centrifugation, washed with water and ethanol, and dried in a vacuum oven at 80 ◦ C overnight to obtain MoS2 –graphene nanocomposites.
2.4. Preparation of AuNPs The preparation of AuNPs followed Frens’ method [34]. Briefly, 100 mL 0.01% (w/v) HAuCl4 solution was brought to vigorous boiling with stirring. Then 0.7 mL 2.0% (w/v) trisodium citrate solution was added to the HAuCl4 solution quickly. The color of the solution changed from pale yellow to wine red in a few seconds. The mixture was maintained at the boiling point for 10 min and then cooled to room temperature under continued stirring. The AuNPs with average size of 16 nm was synthesized and stored at 4 ◦ C.
2.5. Fabrication of the aptasensor The fabrication of the aptasensor was shown in Scheme 1. First of all, the GCE was polished with 0.3 and 0.05 m alumina powder sequentially. After a short rinse and sonication in ethanol and water, the GCE was to be dried. In order to fabricate the aptasensor, 10 L 1 mg mL−1 of MoS2 –graphene suspension was dropped on the pretreated GCE which then was dried. Then, 6 L 0.3 wt% Nafion solution and 6 L 10 mM Ru(bpy)3 2+ solution were cast on the MoS2 –graphene/GCE to obtain Nafion/MoS2 –graphene/GCE and successively Ru(bpy)3 2+ /Nafion/MoS2 –graphene/GCE, respectively. After that, 6 L AuNPs colloids were modified on the electrode to prepare AuNPs/Ru(bpy)3 2+ /Nafion/MoS2 –graphene/GCE. And the resultant electrode was immersed into 1.0 M SH-aptamer solution for 12 h to form the aptasensor through strong Au S bond. The unbonded aptamer was removed by washing the modified electrode with PBS. At last the aptasensor was incubated with 10 L 1% BSA for 1 h in order to block nonspecific binding sites, followed by washing with Tris–HCl buffer and stored at 4 ◦ C. Before the aptasensor was used to detect target thrombin, it was immersed into thrombin solution at 37 ◦ C for 1 h.
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Scheme 1. The schematic representation of the fabrication process for ECL aptasensor.
2.6. ECL measurement ECL intensity of the aptasensor was measured with a BPCL UltraWeak Luminescence Analyzer (Institute of Biophysics, Chinese Academy of Science, Beijing, China) with a CR 120 type photomultiplier tube (Binsong Photonics, Beijing, China). CV mode with continuous potential scanning from 0.2 to 1.25 V and scanning rate of 100 mV s−1 were applied to achieve ECL signal in 5 mL detection solution (pH 7.4, 0.1 M PBS containing 10.0 M TPA as coreactant). The 0.1 M PBS was prepared by mixing K2 HPO4 , NaH2 PO4 and KCl together. The ECL and CV curves were recorded simultaneously.
characteristic peaks at 450 cm−1 can be attributed to the Mo S vibration in MoS2 –graphene nanocomposites. To verify the presence of MoS2 on graphene, the XPS measurements were carried out. As shown in Fig. 3A, one peak at 284.7 eV in the C 1s spectrum is ascribed to C C of graphene. In Fig. 3B, the peaks at about 162.2 and 163.4 eV are related to S 2p 3/2 and S 2p 1/2 binding energy [37], respectively. In addition, two strong peaks in Fig. 3C are observed at approximately 229.5 and 232.9 eV which could be attributed to Mo 3d 5/2 and Mo 3d 3/2 binding energy [38], respectively. The results confirm that the MoS2 –graphene nanocomposites have been prepared successfully. 3.2. Electrochemical behavior of the aptasensor
3. Results and discussion 3.1. Characterization of MoS2 –graphene nanocomposites MoS2 –graphene nanocomposites were characterized by SEM and TEM, and the corresponding images were shown in Fig. 1. The SEM image in Fig. 1A reveals that the MoS2 –graphene nanocomposites are 3D flowerlike multilayer. The 3D structure might be caused by the self-assembly of in situ reduced flexible GO into the layered structure of MoS2 through partial overlapping or coalescing during the hydrothermal process [35]. This 3D structure could facilitate the electrons transfer of electrode reaction and also enhance the stability of the MoS2 –graphene composites due to the – interaction between graphene and GCE [36]. From the TEM image in Fig. 1B, the layered MoS2 on the graphene surface can be seen clearly. The high resolution TEM (HRTEM) image of MoS2 –graphene composites in Fig. 1C shows the layered MoS2 with about 5–12 layers and an interlayer distance of 1 nm. A selected-area electron diffraction pattern shown in Fig. 1D indicates that the graphene layers do not stack together. The MoS2 –graphene nanocomposites were further characterized by XRD and FTIR as shown in Fig. 2. XRD pattern of the MoS2 –graphene composites shows (0 0 2) diffraction peak at 2 = 8.64◦ (Fig. 2A) with d = 1 nm (calculated by Bragg equation), demonstrating its multilayer structure and consistent with the result from HRTEM. FTIR spectrum (Fig. 2B) displays the characteristic absorption bonds of O H stretching vibration of carboxyl group at 3421 cm−1 and C O stretching vibration at 1640 cm−1 . The peak around 1387 cm−1 is the vibration of C C bond. The
CV is an effective method for probing the process of electrode modification. Fig. 4A shows the CV curves of the stepwise modified electrode using Fe(CN)6 3− /Fe(CN)6 4− as redox probe. A well-defined redox peak of Fe(CN)6 3− /Fe(CN)6 4− was observed at the bare GCE (curve a). When MoS2 –graphene nanocomposites were coated on the electrode, the peak current markedly increased due to the excellent conductivity of MoS2 –graphene composites (curve b). However, the peak current decreased after Nafion was cast because the modification of Nafion hindered the transfer of the electrons (curve c). The peak current further decreased after the electrode was modified with Ru(bpy)3 2+ (curve d). When AuNPs were combined with the immobilized Ru(bpy)3 2+ , the peak current increased significantly (curve e) because of the good conductivity of AuNPs. Whereas, the electrode modified with aptamer exhibited a decrease peak current (curve f). This is resulted from the nonconductive property of aptamer. While the prepared electrode was blocked with BSA (curve g) and incubated with thrombin (curve h), the CV responses declined in succession as the hindrance caused by BSA and thrombin. EIS was also used to study the interface properties of modified electrodes. The semi-circle diameter equals to the value of electron-transfer resistance (Ret ) which controls the electron-transfer kinetics of the redox probe at the electrode interface. As shown in Fig. 4B, the bare GCE shows a small semi-circle (curve a). When MoS2 –graphene nanocomposites were coated on the electrode, a decrease of Ret value (curve b) could be observed. Then the Ret value increased on Nafion modified MoS2 –graphene/GCE (curve c) and further on
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Fig. 1. SEM (A), TEM (B) and HRTEM (C) images of MoS2 –graphene nanocomposites. The electron diffraction pattern of MoS2 –graphene (D).
Fig. 2. XRD (A) and FTIR spectrum (B) of MoS2 –graphene.
Fig. 3. C 1s (A), S 2p (B) and Mo 3d (C) peaks in XPS spectra of MoS2 –graphene.
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Fig. 4. CV (A), EIS (B) and ECL (C) behaviors of bare GCE (a), MoS2 –graphene/GCE (b), Nafion/MoS2 –graphene/GCE (c), Ru(bpy)3 2+ /Nafion/MoS2 –graphene/GCE (d), AuNPs/Ru(bpy)3 2+ /Nafion/MoS2 –graphene/GCE (e), SH-aptamer/AuNPs/Ru(bpy)3 2+ /Nafion/MoS2 –graphene/GCE (f), BSA/SH-aptamer/AuNPs/Ru(bpy)3 2+ /Nafion/MoS2 – graphene/GCE (g) and Thrombin/BSA/SH-aptamer/AuNPs/Ru(bpy)3 2+ /Nafion/MoS2 –graphene/GCE (h). CV and EIS in 10 mM Fe(CN)6 3− /Fe(CN)6 4− (1:1) containing 0.1 M KCl. The CVs proceeded between −0.2 and 0.6 V with a scan rate of 100 mV s−1 . (D) ECL curves in the presence (e) and absence (e ) of MoS2 –graphene. The working solution was 0.1 M pH 7.4 PBS containing 0.1 M KCl and 10.0 M TPA. The CVs proceeded between 0.2 and 1.25 V with a scan rate of 100 mV s−1 .
Ru(bpy)3 2+ /Nafion/MoS2 –graphene/GCE (curve d). With further modification by AuNPs, the Ret value decreased significantly (curve e), which is attributed to the good conductivity of AuNPs. However, when SH-aptamer immobilized on the modified electrode, an enhancement of Ret value was occurred (curve f), which demonstrated the aptamer had been successfully immobilized on the electrode. And the Ret value further increased after BSA (curve g) and thrombin (curve h) were successively adsorbed onto the electrode surface. The results obtained from EIS are in well agreement with the results of CV, all demonstrating that the aptasensor is constructed successfully. 3.3. ECL behavior of the aptasensor ECL behavior of the aptasensor at each modified stage was tested and the results were illustrated in Fig. 4C. The bare GCE has no ECL signal (curve a). With only MoS2 –graphene nanocomposites (curve b) and Nafion (curve c) on the GCE surface, almost no ECL signal was observed because of the lack of luminescence reagent. When Ru(bpy)3 2+ was immobilized on the electrode, an obvious ECL signal appeared (curve d). Furthermore, as AuNPs was modified on Ru(bpy)3 2+ /Nafion/MoS2 –graphene/GCE, an enhanced ECL signal could be observed (curve e). However, the ECL signal lowered with the combination of aptamer (curve f). The ECL intensity further decreased after addition of BSA (curve g) and thrombin (curve h), respectively. In order to estimate the effect of the MoS2 –graphene nanocomposites on ECL signal, two sensors, AuNPs/Ru(bpy)3 2+ / Nafion/MoS2 –graphene/GCE (curve e in Fig. 4D) and AuNPs/Ru(bpy)3 2+ /Nafion/GCE (curve e ), were fabricated and compared under the same experimental conditions. The results reveal that the ECL intensity of sensor with MoS2 –graphene is much higher than that without the nanocomposites, indicating that MoS2 –graphene plays an important role in the aptasensor.
3.4. Analytical performance of the ECL aptasensor In this work, the quantification of thrombin is based on the decrease of ECL response of the aptasensor. The ECL intensity (I) changes linearly with the logarithm of thrombin concentration (−log c) in the range of 1.0 × 10−14 to 5.0 × 10−9 M with a regression equation of I = −8958.6 − 1261.9 log c (R = 0.995), as shown in Fig. 5A. The detection limit of thrombin was calculated to be 3.6 × 10−15 M (S/N = 3). According to the report of Nair and Alam [39], the diffusion-limited transport of DNA through water molecules dictates sensor response which varies as ∼t1/2 for a 1D planar sensor and ∼t1 for 2D and 3D sensors. In our work, the sensor was constructed by 3D structured MoS2 –graphene and AuNPs. Therefore, the diffusion transport on our sensor should increase faster with the increasing of settling time compared with that on planar sensor in other study [40], even it used preconcentration technique to increase the settling time. Such a fast diffusion transport results in a fast capture dynamics of biomolecules and therefore the low detection limit (fM level) in short settling time. This is in accordance with the Nair and Alam’s report, they validated that 2D and 3D sensors can detect down to 100 fM concentration. The stability of the aptasensor was also evaluated under consecutive cyclic scans for 35 cycles (Fig. 5B). The RSD for the ECL intensity of 35 cycles was 2.5%, indicating the good stability of the aptasensor. 3.5. Selectivity of the ECL aptasensor The selectivity of this aptasensor was assessed by interference experiments. Six different samples (1.0 nM thrombin, 1 M HSA, 1 M hIgG, 1.0 nM thrombin + 1 M HSA, 1.0 nM thrombin + 1 M hIgG, and 1.0 nM thrombin + 1 M HSA + 1 M hIgG) were detected separately. The results in Fig. 6 show that HSA and hIgG have negligible interference on the detection of thrombin, indicating the good selectivity of the aptasensor.
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Fig. 5. The calibration curve of thrombin detection (A) and ECL intensity-time curve (B) under continuous CVs for 35 cycles. Other conditions for ECL measurement are the same as Fig. 4D.
4. Conclusions In summary, a label-free Ru(bpy)3 2+ ECL aptasensor for ultrasensitive and highly selective detection of thrombin was successfully fabricated through a layer-by-layer assembling strategy. MoS2 –graphene nanocomposites as matrix immobilized on GCE could improve the conductivity and loading capacity of the sensing interface. AuNPs were served as both the ECL signal amplification reagent and the immobilization platform for aptamer. Based on the present strategy, thrombin as a model protein could be detected directly by the change of ECL signal without the introduction of additional probe. The applicability of the aptasensor was demonstrated in the determination of human plasma samples. This method provides a new approach for highly sensitive and specific detection of disease-related proteins in complex biological samples. Fig. 6. Specificity of the ECL aptasensor. The concentration of thrombin, HSA and IgG is 1.0 nM, 1.0 M and 100 M. Other conditions for ECL measurement are the same as Fig. 4D.
Table 1 Analytical results of thrombin in human plasma samples. Sample of traumatic injury patients
Found (×10−14 M)
Sample of non-traumatic injury patients
Found (×10−14 M)
1 2 3
560 200 680
4 5 6
5.5 5.0 4.6
3.6. Application of the ECL aptasensor The application of the present protocol was demonstrated by analyzing thrombin in six human plasma samples provided by Xinyang Central Hospital. The six human plasma samples were divided into two groups, three traumatic injury patients (Nos. 1–3) and three non-traumatic injury patients (Nos. 4–6). The analytical results were listed in Table 1. It can be seen that the thrombin found in the three plasma samples of traumatic injury patients is much higher than that in the non-traumatic injury patients. In addition, the credibility of the aptasensor was further investigated by using the standard addition method. The recoveries of thrombin in the plasma samples at three different spiked concentration levels (1.0 × 10−13 , 5.0 × 10−12 and 1.0 × 10−10 M) were found to be in the range of 88.6–105.0%, and the RSD values were lower than 3.7%. The results suggest that the proposed aptasensor is suitable for the determination of thrombin in real biosamples.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Grants 21375114 and U1304214), the Project of Science and Technology Development of Henan Province (142300410197), and the Foundation of Henan Educational Committee (14A150013).
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
A novel label-free electrochemiluminescence aptasensor based on layered flowerlike molybdenum sulfide–graphene nanocomposites as matrix Yan-Ming Liu a,∗ , Gui-Fang Shi a , Jing-Jing Zhang a , Min Zhou a , Jun-Tao Cao a , Ke-Jing Huang a , Shu-Wei Ren b a b
College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000, China Xinyang Central Hospital, Xinyang 464000, China
a r t i c l e
i n f o
Article history: Received 24 March 2014 Received in revised form 2 July 2014 Accepted 8 July 2014 Available online 16 July 2014 Keywords: Molybdenum sulfide–graphene nanocomposites Electrochemiluminescence Thrombin Aptasensor
a b s t r a c t A label-free and ultrasensitive electrochemiluminescence (ECL) aptasensor was constructed for the detection of thrombin. Molybdenum sulfide–graphene nanocomposites with good conductivity and large surface area were immobilized on glassy carbon electrode (GCE), and then Nafion was fixed to chemosorb the Ru(bpy)3 2+ used as luminescence agent. Subsequently, gold nanoparticles (AuNPs) were modified on the electrode to immobilize the thiol-modified thrombin aptamer for fabrication of the thrombin aptasensor. The proposed ECL aptasensor produced the ultrasensitive detection of thrombin with a low detection limit of 3.6 × 10−15 M (S/N = 3) and over a wide target concentration range from 1.0 × 10−14 to 5.0 × 10−9 M. The aptasensor has been successfully applied in the determination of thrombin in human plasma samples of both traumatic and non-traumatic injury patients, indicating its promise in biochemical analysis. The recoveries of thrombin in human plasma samples are between 88.6% and 105.0%, and the RSD values are no more than 3.7%. The results demonstrate that this aptasensor has excellent sensitivity, selectivity and stability. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Thrombin is a kind of extracellular serine protease and involved in a number of physiological and pathological processes, such as blood coagulation, thrombosis, cardiovascular diseases, platelet activation, and even in the angiogenesis as a biomarker [1]. Thrombin of low level in organism is neuroprotective while high level is deleterious. Therefore, the determination of thrombin is very important in biochemical analysis. Aptamer as a novel capture agent has been intensively used for protein detection with the advantages of high specificity, affinity, nontoxicity, good stability, and relatively easy preparation [2,3]. Furthermore, aptamer can be easily modified with a variety of chemical groups that can provide an extraordinary flexibility in different assays and can be fixed on electrode surface through various chemical forces [4,5]. AuNPs have attracted great attentions due to their good biocompatibility, excellent conductivity and large specific surface area. The conjugates of AuNPs and thiol-modified aptamer through an Au S bond have
∗ Corresponding author. Fax: +86 376 6392889. E-mail address: [email protected] (Y.-M. Liu). http://dx.doi.org/10.1016/j.colsurfb.2014.07.011 0927-7765/© 2014 Elsevier B.V. All rights reserved.
been widely studied [6]. Up to now, various aptamer-based methods for thrombin detection have been reported, such as enhanced surface plasmon resonance [7], chronocoulometry [8], fluorescence [9], differential pulse voltammetry [10,11]. Electrochemiluminescence (ECL) has become an attractive and powerful detection tool because of its high sensitivity, good selectivity, wide linear range, and low equipment cost [12,13]. Ru(bpy)3 2+ ECL system has been successfully used in the analysis of amino acids [14], drugs [15], DNA [16] and protein [17,18] due to its excellent stability and high ECL quantum yield. The immobilization of Ru(bpy)3 2+ on electrode surface could reduce the amount of the reagent and simplify the experimental design. However, the leakage of Ru(bpy)3 2+ , resulting from its inherent water-solubility, always hinders the gain of stable ECL sensor. So far various methods for immobilization Ru(bpy)3 2+ have been developed to solve this problem [19–21]. For example, Ru(bpy)3 2+ -doped silica nanoparticles were applied to develop an aptamer-based ECL sensor for thrombin detection with a detection limit of 2.0 × 10−13 M in Lin’s work [19]. Zhang et al. [20] developed a signal-on junction-probe ECL biosensor for the detection of thrombin using AuNPs-Ru(bpy)3 2+ as signal probe. Fang et al. [21] utilized aptamer functionalized Ru(bpy)3 2+ as labels to probe the
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thrombin with the detection limit of 10 nM. In these methods, a time-consuming labeling process was involved, so novel label free Ru(bpy)3 2+ ECL biosensors are highly desired. Recently, 2-dimensional layered materials such as MoS2 , SnS2 and WS2 have been widely used as lubricants, catalysts [22], electrode materials for capacitors [23], and lithium-ion batteries [24] due to their high surface area, excellent chemical stability and strong mechanical strength. MoS2 is a family member of transition-metal dichalcogenides and is composed of Mo metal layers sandwiched between two sulfur layers [25,26]. Its layered structure is therefore expected to act as an excellent functional material because the 2-dimensional electron–electron correlations among Mo atoms would aid in enhancing planar electric transportation properties. However, few attentions have been paid on the analytical application of MoS2 as an electrode material for ECL sensor because its electrical conductivity is lower than that of carbon nanotube/graphene [27]. Graphene has excellent electrical conductivity, large surface area and chemical stability [3,28]. It has been applied in the detection of ATP with detection limit of 2.01 × 10−11 M [29]. Graphene-based nanomaterials have been extensively studied [30]. The combination of graphene and MoS2 may provide a new nanomaterial with good electrochemical properties. Our group prepared an electrochemical sensor based on MoS2 –graphene nanohybrid for acetaminophen detection with detection limit of 2.0 × 10−8 M [31]. In our previous work, we have developed a CdSe-ZnS quantum dots labeled, sandwich-type ECL aptasensor for thrombin detection [32]. CdSe-ZnS quantum dots were used as ECL emission reagent and two aptamers were used to form a sandwich-type structure. In this work, we fabricated a simple, label-free single chain aptamer aptasensor used Ru(bpy)3 2+ as the ECL reagent, and MoS2 –graphene nanocomposites with flowerlike structure as matrix to immobilize Ru(bpy)3 2+ . The thrombin was selected as model analyte. The process of the stepwise modification of GCE was investigated in detail. The high sensitive detection of thrombin in complex samples was achieved, demonstrating the great potential of the ECL aptasensor for protein detection. 2. Experimental 2.1. Reagents Ru(bpy)3 Cl2 ·6H2 O and HAuCl4 ·3H2 O were purchased from Alfa Aesar (Tianjin, China). Na2 MoO4 ·2H2 O, l-cysteine, tri-npropylamine (TPA, 98%), and citric acid trisodium salt dihydrate (C6 H5 Na3 O7 ·2H2 O) was from the Sinopharm Group Chemical Reagent Co. Ltd. (Shanghai, China). SH-aptamer (5 -SH-(CH2 )6 -TTT TTT TTT TTT TTT GGT TGG TGT GGT TGG-3 ) was synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). Human thrombin, human immunoglobulin (hIgG), human serum albumin (HSA), and bovine serum albumin (BSA, Mr = 67,000) from Shanghai Solarbio Bioscience & Technology Co. Ltd. (Seebio Biotechnology). Nafion (5 wt%) from Sigma–Aldrich (USA). Tris–HCl buffer (pH 7.4) containing 20 mM Tris, 100 mM NaCl, 5 mM KCl, 1 mM MgCl2 and 1 mM CaCl2 was used to dissolve thrombin. 0.2 M phosphate buffer saline (PBS, pH 7.4) from Sangon Biotech Co. Ltd. (Shanghai, China) was used to dissolve aptamer. All chemicals were of analytical grade. The water used was processed with an Ultrapure Water System (Kangning Water Treatment Solution Provider, China). 2.2. Apparatus Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements were carried out with RST5200
electrochemical workstation (Zhengzhou Shiruisi Technology Co. Ltd., China). A three-electrode system was composed of a modified GCE (ϕ = 3 mm) as the working electrode, a Pt spiral wire as the counter electrode and an Ag/AgCl as the reference electrode. Transmission electron microscope (TEM) images were recorded by JEM-2100F (JEOL Ltd., Tokyo, Japan) TEM system. Scanning electron microscopy (SEM) images were obtained by S-4800 (Hitachi, Tokyo, Japan). X-ray powder diffraction (XRD) pattern was operated on RigakuD/Maxr-A X-ray diffractometer (Japan). X-ray photoelectron spectroscopy (XPS) measurement was performed by Kratos Axis Ultra electron spectrometer (UK). Fourier transform infrared (FTIR) spectrum was recorded on Bruker TENZOR 27 spectrophotometer (Germany).
2.3. Preparation of MoS2 –graphene nanocomposites The graphene oxide (GO) was synthesized from graphite flake using the Hummers’ method [33]. The preparation of MoS2 –graphene nanocomposites followed the procedure of our previous work [31]. Briefly, 0.1 g GO and 50 mL water were mixed and treated with ultrasonication. Then, 0.5 g Na2 MoO4 ·2H2 O was added and ultrasonicated for 20 min and the pH value was adjusted to 6.5 with 0.1 M NaOH. Subsequently, 1.0 g l-cysteine and 40 mL water were added and then transferred into a Teflon-lined stainless steel autoclave which was heated to 180 ◦ C for 48 h. After cooling naturally, the black precipitates was collected by centrifugation, washed with water and ethanol, and dried in a vacuum oven at 80 ◦ C overnight to obtain MoS2 –graphene nanocomposites.
2.4. Preparation of AuNPs The preparation of AuNPs followed Frens’ method [34]. Briefly, 100 mL 0.01% (w/v) HAuCl4 solution was brought to vigorous boiling with stirring. Then 0.7 mL 2.0% (w/v) trisodium citrate solution was added to the HAuCl4 solution quickly. The color of the solution changed from pale yellow to wine red in a few seconds. The mixture was maintained at the boiling point for 10 min and then cooled to room temperature under continued stirring. The AuNPs with average size of 16 nm was synthesized and stored at 4 ◦ C.
2.5. Fabrication of the aptasensor The fabrication of the aptasensor was shown in Scheme 1. First of all, the GCE was polished with 0.3 and 0.05 m alumina powder sequentially. After a short rinse and sonication in ethanol and water, the GCE was to be dried. In order to fabricate the aptasensor, 10 L 1 mg mL−1 of MoS2 –graphene suspension was dropped on the pretreated GCE which then was dried. Then, 6 L 0.3 wt% Nafion solution and 6 L 10 mM Ru(bpy)3 2+ solution were cast on the MoS2 –graphene/GCE to obtain Nafion/MoS2 –graphene/GCE and successively Ru(bpy)3 2+ /Nafion/MoS2 –graphene/GCE, respectively. After that, 6 L AuNPs colloids were modified on the electrode to prepare AuNPs/Ru(bpy)3 2+ /Nafion/MoS2 –graphene/GCE. And the resultant electrode was immersed into 1.0 M SH-aptamer solution for 12 h to form the aptasensor through strong Au S bond. The unbonded aptamer was removed by washing the modified electrode with PBS. At last the aptasensor was incubated with 10 L 1% BSA for 1 h in order to block nonspecific binding sites, followed by washing with Tris–HCl buffer and stored at 4 ◦ C. Before the aptasensor was used to detect target thrombin, it was immersed into thrombin solution at 37 ◦ C for 1 h.
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Scheme 1. The schematic representation of the fabrication process for ECL aptasensor.
2.6. ECL measurement ECL intensity of the aptasensor was measured with a BPCL UltraWeak Luminescence Analyzer (Institute of Biophysics, Chinese Academy of Science, Beijing, China) with a CR 120 type photomultiplier tube (Binsong Photonics, Beijing, China). CV mode with continuous potential scanning from 0.2 to 1.25 V and scanning rate of 100 mV s−1 were applied to achieve ECL signal in 5 mL detection solution (pH 7.4, 0.1 M PBS containing 10.0 M TPA as coreactant). The 0.1 M PBS was prepared by mixing K2 HPO4 , NaH2 PO4 and KCl together. The ECL and CV curves were recorded simultaneously.
characteristic peaks at 450 cm−1 can be attributed to the Mo S vibration in MoS2 –graphene nanocomposites. To verify the presence of MoS2 on graphene, the XPS measurements were carried out. As shown in Fig. 3A, one peak at 284.7 eV in the C 1s spectrum is ascribed to C C of graphene. In Fig. 3B, the peaks at about 162.2 and 163.4 eV are related to S 2p 3/2 and S 2p 1/2 binding energy [37], respectively. In addition, two strong peaks in Fig. 3C are observed at approximately 229.5 and 232.9 eV which could be attributed to Mo 3d 5/2 and Mo 3d 3/2 binding energy [38], respectively. The results confirm that the MoS2 –graphene nanocomposites have been prepared successfully. 3.2. Electrochemical behavior of the aptasensor
3. Results and discussion 3.1. Characterization of MoS2 –graphene nanocomposites MoS2 –graphene nanocomposites were characterized by SEM and TEM, and the corresponding images were shown in Fig. 1. The SEM image in Fig. 1A reveals that the MoS2 –graphene nanocomposites are 3D flowerlike multilayer. The 3D structure might be caused by the self-assembly of in situ reduced flexible GO into the layered structure of MoS2 through partial overlapping or coalescing during the hydrothermal process [35]. This 3D structure could facilitate the electrons transfer of electrode reaction and also enhance the stability of the MoS2 –graphene composites due to the – interaction between graphene and GCE [36]. From the TEM image in Fig. 1B, the layered MoS2 on the graphene surface can be seen clearly. The high resolution TEM (HRTEM) image of MoS2 –graphene composites in Fig. 1C shows the layered MoS2 with about 5–12 layers and an interlayer distance of 1 nm. A selected-area electron diffraction pattern shown in Fig. 1D indicates that the graphene layers do not stack together. The MoS2 –graphene nanocomposites were further characterized by XRD and FTIR as shown in Fig. 2. XRD pattern of the MoS2 –graphene composites shows (0 0 2) diffraction peak at 2 = 8.64◦ (Fig. 2A) with d = 1 nm (calculated by Bragg equation), demonstrating its multilayer structure and consistent with the result from HRTEM. FTIR spectrum (Fig. 2B) displays the characteristic absorption bonds of O H stretching vibration of carboxyl group at 3421 cm−1 and C O stretching vibration at 1640 cm−1 . The peak around 1387 cm−1 is the vibration of C C bond. The
CV is an effective method for probing the process of electrode modification. Fig. 4A shows the CV curves of the stepwise modified electrode using Fe(CN)6 3− /Fe(CN)6 4− as redox probe. A well-defined redox peak of Fe(CN)6 3− /Fe(CN)6 4− was observed at the bare GCE (curve a). When MoS2 –graphene nanocomposites were coated on the electrode, the peak current markedly increased due to the excellent conductivity of MoS2 –graphene composites (curve b). However, the peak current decreased after Nafion was cast because the modification of Nafion hindered the transfer of the electrons (curve c). The peak current further decreased after the electrode was modified with Ru(bpy)3 2+ (curve d). When AuNPs were combined with the immobilized Ru(bpy)3 2+ , the peak current increased significantly (curve e) because of the good conductivity of AuNPs. Whereas, the electrode modified with aptamer exhibited a decrease peak current (curve f). This is resulted from the nonconductive property of aptamer. While the prepared electrode was blocked with BSA (curve g) and incubated with thrombin (curve h), the CV responses declined in succession as the hindrance caused by BSA and thrombin. EIS was also used to study the interface properties of modified electrodes. The semi-circle diameter equals to the value of electron-transfer resistance (Ret ) which controls the electron-transfer kinetics of the redox probe at the electrode interface. As shown in Fig. 4B, the bare GCE shows a small semi-circle (curve a). When MoS2 –graphene nanocomposites were coated on the electrode, a decrease of Ret value (curve b) could be observed. Then the Ret value increased on Nafion modified MoS2 –graphene/GCE (curve c) and further on
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Fig. 1. SEM (A), TEM (B) and HRTEM (C) images of MoS2 –graphene nanocomposites. The electron diffraction pattern of MoS2 –graphene (D).
Fig. 2. XRD (A) and FTIR spectrum (B) of MoS2 –graphene.
Fig. 3. C 1s (A), S 2p (B) and Mo 3d (C) peaks in XPS spectra of MoS2 –graphene.
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Fig. 4. CV (A), EIS (B) and ECL (C) behaviors of bare GCE (a), MoS2 –graphene/GCE (b), Nafion/MoS2 –graphene/GCE (c), Ru(bpy)3 2+ /Nafion/MoS2 –graphene/GCE (d), AuNPs/Ru(bpy)3 2+ /Nafion/MoS2 –graphene/GCE (e), SH-aptamer/AuNPs/Ru(bpy)3 2+ /Nafion/MoS2 –graphene/GCE (f), BSA/SH-aptamer/AuNPs/Ru(bpy)3 2+ /Nafion/MoS2 – graphene/GCE (g) and Thrombin/BSA/SH-aptamer/AuNPs/Ru(bpy)3 2+ /Nafion/MoS2 –graphene/GCE (h). CV and EIS in 10 mM Fe(CN)6 3− /Fe(CN)6 4− (1:1) containing 0.1 M KCl. The CVs proceeded between −0.2 and 0.6 V with a scan rate of 100 mV s−1 . (D) ECL curves in the presence (e) and absence (e ) of MoS2 –graphene. The working solution was 0.1 M pH 7.4 PBS containing 0.1 M KCl and 10.0 M TPA. The CVs proceeded between 0.2 and 1.25 V with a scan rate of 100 mV s−1 .
Ru(bpy)3 2+ /Nafion/MoS2 –graphene/GCE (curve d). With further modification by AuNPs, the Ret value decreased significantly (curve e), which is attributed to the good conductivity of AuNPs. However, when SH-aptamer immobilized on the modified electrode, an enhancement of Ret value was occurred (curve f), which demonstrated the aptamer had been successfully immobilized on the electrode. And the Ret value further increased after BSA (curve g) and thrombin (curve h) were successively adsorbed onto the electrode surface. The results obtained from EIS are in well agreement with the results of CV, all demonstrating that the aptasensor is constructed successfully. 3.3. ECL behavior of the aptasensor ECL behavior of the aptasensor at each modified stage was tested and the results were illustrated in Fig. 4C. The bare GCE has no ECL signal (curve a). With only MoS2 –graphene nanocomposites (curve b) and Nafion (curve c) on the GCE surface, almost no ECL signal was observed because of the lack of luminescence reagent. When Ru(bpy)3 2+ was immobilized on the electrode, an obvious ECL signal appeared (curve d). Furthermore, as AuNPs was modified on Ru(bpy)3 2+ /Nafion/MoS2 –graphene/GCE, an enhanced ECL signal could be observed (curve e). However, the ECL signal lowered with the combination of aptamer (curve f). The ECL intensity further decreased after addition of BSA (curve g) and thrombin (curve h), respectively. In order to estimate the effect of the MoS2 –graphene nanocomposites on ECL signal, two sensors, AuNPs/Ru(bpy)3 2+ / Nafion/MoS2 –graphene/GCE (curve e in Fig. 4D) and AuNPs/Ru(bpy)3 2+ /Nafion/GCE (curve e ), were fabricated and compared under the same experimental conditions. The results reveal that the ECL intensity of sensor with MoS2 –graphene is much higher than that without the nanocomposites, indicating that MoS2 –graphene plays an important role in the aptasensor.
3.4. Analytical performance of the ECL aptasensor In this work, the quantification of thrombin is based on the decrease of ECL response of the aptasensor. The ECL intensity (I) changes linearly with the logarithm of thrombin concentration (−log c) in the range of 1.0 × 10−14 to 5.0 × 10−9 M with a regression equation of I = −8958.6 − 1261.9 log c (R = 0.995), as shown in Fig. 5A. The detection limit of thrombin was calculated to be 3.6 × 10−15 M (S/N = 3). According to the report of Nair and Alam [39], the diffusion-limited transport of DNA through water molecules dictates sensor response which varies as ∼t1/2 for a 1D planar sensor and ∼t1 for 2D and 3D sensors. In our work, the sensor was constructed by 3D structured MoS2 –graphene and AuNPs. Therefore, the diffusion transport on our sensor should increase faster with the increasing of settling time compared with that on planar sensor in other study [40], even it used preconcentration technique to increase the settling time. Such a fast diffusion transport results in a fast capture dynamics of biomolecules and therefore the low detection limit (fM level) in short settling time. This is in accordance with the Nair and Alam’s report, they validated that 2D and 3D sensors can detect down to 100 fM concentration. The stability of the aptasensor was also evaluated under consecutive cyclic scans for 35 cycles (Fig. 5B). The RSD for the ECL intensity of 35 cycles was 2.5%, indicating the good stability of the aptasensor. 3.5. Selectivity of the ECL aptasensor The selectivity of this aptasensor was assessed by interference experiments. Six different samples (1.0 nM thrombin, 1 M HSA, 1 M hIgG, 1.0 nM thrombin + 1 M HSA, 1.0 nM thrombin + 1 M hIgG, and 1.0 nM thrombin + 1 M HSA + 1 M hIgG) were detected separately. The results in Fig. 6 show that HSA and hIgG have negligible interference on the detection of thrombin, indicating the good selectivity of the aptasensor.
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Fig. 5. The calibration curve of thrombin detection (A) and ECL intensity-time curve (B) under continuous CVs for 35 cycles. Other conditions for ECL measurement are the same as Fig. 4D.
4. Conclusions In summary, a label-free Ru(bpy)3 2+ ECL aptasensor for ultrasensitive and highly selective detection of thrombin was successfully fabricated through a layer-by-layer assembling strategy. MoS2 –graphene nanocomposites as matrix immobilized on GCE could improve the conductivity and loading capacity of the sensing interface. AuNPs were served as both the ECL signal amplification reagent and the immobilization platform for aptamer. Based on the present strategy, thrombin as a model protein could be detected directly by the change of ECL signal without the introduction of additional probe. The applicability of the aptasensor was demonstrated in the determination of human plasma samples. This method provides a new approach for highly sensitive and specific detection of disease-related proteins in complex biological samples. Fig. 6. Specificity of the ECL aptasensor. The concentration of thrombin, HSA and IgG is 1.0 nM, 1.0 M and 100 M. Other conditions for ECL measurement are the same as Fig. 4D.
Table 1 Analytical results of thrombin in human plasma samples. Sample of traumatic injury patients
Found (×10−14 M)
Sample of non-traumatic injury patients
Found (×10−14 M)
1 2 3
560 200 680
4 5 6
5.5 5.0 4.6
3.6. Application of the ECL aptasensor The application of the present protocol was demonstrated by analyzing thrombin in six human plasma samples provided by Xinyang Central Hospital. The six human plasma samples were divided into two groups, three traumatic injury patients (Nos. 1–3) and three non-traumatic injury patients (Nos. 4–6). The analytical results were listed in Table 1. It can be seen that the thrombin found in the three plasma samples of traumatic injury patients is much higher than that in the non-traumatic injury patients. In addition, the credibility of the aptasensor was further investigated by using the standard addition method. The recoveries of thrombin in the plasma samples at three different spiked concentration levels (1.0 × 10−13 , 5.0 × 10−12 and 1.0 × 10−10 M) were found to be in the range of 88.6–105.0%, and the RSD values were lower than 3.7%. The results suggest that the proposed aptasensor is suitable for the determination of thrombin in real biosamples.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Grants 21375114 and U1304214), the Project of Science and Technology Development of Henan Province (142300410197), and the Foundation of Henan Educational Committee (14A150013).
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