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An amplified electrochemical aptasensor for thrombin detection based on pseudobienzymic Fe3O4–Au nanocomposites and electroactive hemin/G-quadruplex as signal enhancers† Pei Jing, Wenju Xu,* Huayu Yi, Yongmei Wu, Lijuan Bai and Ruo Yuan* A sensitive and selective electrochemical aptasensor for thrombin detection was constructed based on hemin/G-quadruplex as the signal label and Fe3O4–Au nanocomposites with glucose oxidase (GOx-) and peroxide-mimicking enzyme activity as the signal enhancers. Due to their large surface area and good biocompatibility, Fe3O4–Au nanocomposites were employed to immobilize electroactive hemin/Gquadruplex, which was formed by the conjugation between a single-stranded guanine-rich nucleic acid and hemin. Based on the GOx-mimicking enzyme activity, Au nanoparticles on the surface of the Fe3O4–Au nanocomposites effectively catalyzed the oxidization of glucose in the presence of dissolved O2, accompanied by the production of H2O2. Both the Fe3O4 cores of Fe3O4–Au nanocomposites and hemin/G-quadruplex with H2O2-mimicking enzyme activity could catalyze the reduction of the
Received 3rd December 2013 Accepted 3rd January 2014
generated H2O2, which promoted the electron transfer of hemin and amplified the electrochemical signal. The proposed electrochemical aptasensor had a wide dynamic linear range of 0.1 pM to 20 nM
DOI: 10.1039/c3an02237d
with a lower detection limit of 0.013 pM, which provided a promising method for a sensitive assay for
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the detection of proteins in electrochemical aptasensors.
Introduction Thrombin (TB), is a kind of serine protease of the blood stream chymotrypsin family, it plays an essential role in the production of insoluble brin through the proteolytic cleavage of soluble brinogen.1 In addition, it is involved in some physiological and pathological processes, including leukemia, arterial thrombosis and liver disease.2 Therefore, highly sensitive and cost-efficient methods for the quantitative detection of TB in clinical diagnosis and treatment are crucial. In recent years, great effort has been made to design aptamer-based assays, such as uorescence,3 chemiluminescence,4 surface plasmon resonance,5 colorimetry,6 quartz crystal microbalance7 and electrochemistry,8–10 for TB detection. Compared with other methods that suffer from the disadvantages of complex operation, large-scale and high-cost instrumentation, the aptamer-based electrochemical method has merits of fast response, high sensitivity, portability, simplicity and inexpensive apparatus,11 and has been widely used in the sensitive detection of TB. In these electrochemical aptasensors, many redox mediator labels, such as methylene blue,12 thionine,13 toluidine blue,14 ferrocene15 and so on, have been used to generate an Education Ministry Key Laboratory on Luminescence and Real-TimeAnalysis, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, People's Republic of China. E-mail: [email protected] † Electronic supplementary 10.1039/c3an02237d
information
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(ESI)
available.
See
DOI:
electrochemical signal. Alternatively, hemin [Fe(III)-protoporphyrin IX], the active cofactor for catalases,16 can also generate an electrochemical signal,17 which makes it possible for hemin to act as an excellent electroactive material for constructing an aptasensor. In addition, hemin can intercalate into a singlestranded guanine-rich nucleic acid with a unique G-quadruplex structure to form a hemin/G-quadruplex structure with hydrogen peroxide (HRP)-mimicking DNAzyme activity.18,19 The hemin/ G-quadruplex biocatalytic structure, which possesses the advantages of inexpensive production, greater stability against hydrolysis, relatively easy labelling and which can greatly enhance the response signal,20,21 has been used to detect metal ions,22,23 proteins,24 DNAs25 and small molecules.26 Recently, electroactive or photoactive molecular labelled nanomaterials have been popularly employed in such aptamerbased detection systems to realize great signal amplication and improve the sensitivity of the method.27–30 Among them, Fe3O4 magnetic nanoparticles (Fe3O4 MNPs) with a unique superparamagnetic activity have been widely exploited in biology and medicine to detect small molecules,31 and to separate and detect proteins.32 In previous research, Fe3O4 MNPs have been generally considered to be biology inactive. However, Wei and coworkers have discovered that Fe3O4 MNPs could exhibit intrinsic peroxidase-mimicking enzyme activity.33 Based on these merits, graphene oxide–Fe3O4 magnetic nanocomposites were employed to detect glucose by using the peroxidase-mimicking enzyme activity
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of Fe3O4 MNPs.34 Lately, He et al. prepared discrete and monodispersed Fe3O4–Au@mesoporous SiO2 (MS) microspheres with high GOx- and peroxidase-mimicking enzyme activities.35 The ultrane (4.2–7.2 nm) Au nanoparticles (AuNPs) on the surface of these nanocomposites, acting as a GOx-mimicking enzyme, can catalyze glucose to gluconolactone coupled with the generation of H2O2 in the presence of the dissolved O2. Then, Fe3O4 acts as a peroxidase-mimicking enzyme and further catalyzes the reduction of the obtained H2O2. Owing to its remarkable pseudobienzyme activity, this kind of nanocomposite could be used to prepare a signal amplied aptasensor for the detection of TB. In this work, we designed a sandwich-type electrochemical aptasensor by utilizing pseudobienzymic Fe3O4–Au nanocomposites and an electroactive hemin/G-quadruplex structure as signal enhancers for the sensitive detection of TB. Fe3O4–Au nanocomposites with a large surface area and pseudobienzyme activity were employed for the immobilization of hemin/Gquadruplex to form hemin/G-quadruplex conjugated Fe3O4–Au nanocomposites (secondary aptamer). The target protein was sandwiched between the primary amido-terminated thrombin aptamer I (NH2–TBA I) and the prepared secondary aptamer. In the presence of glucose, active AuNPs of Fe3O4–Au nanocomposites could oxidize glucose and simultaneously produce H2O2 in the presence of dissolved O2. Furthermore, the Fe3O4 cores of Fe3O4–Au nanocomposites and hemin/G-quadruplex could cooperative to electrocatalyze the reduction of H2O2 and to promote the electron transport of hemin, in order to achieve a signicantly amplied electrochemical signal. The results indicated an effective way to quantitatively detect the target TB, according to the linearity between the logarithm of TB concentration and the current response. This amplied strategy could be applied in the development of a simple and sensitive analytical method for the detection of proteins.
Experimental Chemicals and materials TB, hemin, L-cysteine (L-cys), gold chloride (HAuCl4), bovine serum albumin (BSA) and human IgG were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Trisodium citrate, NaBH4, sodium acetate (NaAc), FeCl3$6H2O and ethylene glycol were obtained from Kelong Chemical Company (Chengdu, China). Glucose was obtained from Sinopharm Chemical Reagent Co. Ltd. (China). The human serum samples were obtained from the Ninth People's Hospital of Chongqing (China). Tris-hydroxymethylaminomethane hydrochloride (Tris) was provided by Roche (Switzerland). Thrombin aptamer (TBA): 50 –NH2–(CH2)6–GGTTGGTGTGGTTGG-30 was purchased from Sangon BiotechCo., Ltd (Shanghai, China). Phosphate buffered solution (PBS) with different pH was prepared using 0.1 M Na2HPO4, 0.1 M KH2PO4 and 0.1 M KCl and served as the working buffer throughout the experiment. Tris–HCl buffer (20 mM, pH 7.4) was prepared with 140 mM NaCl, 5 mM KCl, 1 mM CaCl2 and 1 mM MgCl2, and was used as the aptamer buffer. The prepared solutions were kept at 4 C before use. All the other chemicals used were of analytical grade
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and were used directly as received. Double distilled water was used throughout this work. Apparatus Differential pulse voltammetry measurements (DPVs) and cyclic voltammetry measurements (CVs) were performed with a CHI 660D electrochemical workstation (Shanghai Chenhua Instrument, China). A three electrode system, including a modied glassy carbon electrode (GCE, F ¼ 4 mm) as the working electrode, a platinum wire as the counter electrode, a saturated calomel electrode (SCE) as the reference electrode, was employed for the above electrochemical measurements. The scanning electron micrographs (SEM) were taken with a scanning electron microscope (SEM, S-4800, Hitachi, Tokyo, Japan). Synthesis of Fe3O4–Au nanocomposites Fe3O4–Au nanocomposites were prepared via a hydrothermal method according to the literature with minor revision.36 In brief, 0.70 g of FeCl3$6H2O, 0.28 g of trisodium citrate were rst dispersed in 20 mL of ethylene glycol, aer that 1.2 g of NaAc was added into the resulting solution under continual stirring until the mixture became homogeneous and the colour was yellow. Subsequently, the obtained solution was maintained at 200 C for 10 h in a Teon-lined stainless-steel autoclave. When the autoclave had cooled to room temperature, the resulting precipitate was collected by an external magnetic eld. The black products were washed several times with ethanol and nitrogen-saturated water alternately to remove most of the matrices, including trisodium citrate and ethylene glycol. Finally the particles were dried at 60 C in a vacuum drying oven. The prepared Fe3O4 particles (20 mg) were added to 25 mL of nitrogen-saturated water with sonication for 5 min. Aerwards, 405 mL of HAuCl4 (1%) was added to the above dispersion and magnetically stirred constantly for 30 min. 1 mL of freshly prepared NaBH4 (0.15 M) was added into the above solution under vigorous stirring, and then the mixture was stirred for another 30 min. The resulting product was separated and washed with nitrogen-saturated water several times using a magnet. Lastly, Fe3O4–Au nanocomposites were suspended in 1 mL of PBS (0.1 M, pH 7.0) before use. Synthesis of hemin/G-quadruplex conjugated Fe3O4–Au nanocomposites (secondary aptamer) Based on the conjugation of AuNPs to TBA via the reaction with the amine groups of the aptamer, the secondary aptamer was prepared as follows (Scheme 1B): rstly, 60 mL of NH2–TBA II (100 nM) was injected into the prepared solution and gently stirred for 12 h at 4 C. Secondly, 0.5 mg of hemin was added into the above solution and stirred for 40 min. At this time, the hemin/G-quadruplex biocatalyst structure was obtained. Finally, 200 mL of BSA (w/w, 1%) aqueous solution was added into the obtained mixture and stirred for another 40 min to block the unoccupied active sites of the Fe3O4–Au nanocomposites. Aer centrifugation and washing, the hemin/ G-quadruplex conjugated Fe3O4–Au nanocomposites was Analyst, 2014, 139, 1756–1761 | 1757
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with distilled water to remove physically absorbed species. DPV curves of the proposed aptasensor aer incubation with the secondary aptamer in the absence (I0) and presence (I) of 40 mM glucose in 1 mL of PBS (0.1 M, pH 7.0) are shown in Scheme 1C.
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Experimental measurements
Scheme 1 (A) Schematic illustration of the stepwise fabrication of the electrochemical aptasensor. (B) Procedure for the preparation of hemin/G-quadruplex conjugated Fe3O4–Au nanocomposites (secondary aptamer). (C) DPV cures of the proposed aptasensor after incubation with the secondary aptamer in the absence (I0) and presence (I) of 40 mM glucose in 1 mL of PBS (0.1 M, pH 7.0).
resuspended in 1 mL of Tris–HCl buffer and stored at 4 C for further use. In order to study the amplication properties of the Fe3O4– Au nanocomposites, hemin/G-quadruplex conjugated Au nanocomposites were also prepared in a similar way but without Fe3O4. AuNPs (5 nm) were prepared according to the reported method with some modications by using KBH4 as a reductant and sodium citrate as a stabilizer.36 Briey, 1 mL of HAuCl4 (1%) and 2 mL of sodium citrate (0.03 M) were added to 50 mL of double distilled water and stirred, followed by the addition of 1 mL of freshly prepared NaBH4 (0.1 M). The resulting solution whose colour changed to wine red was maintained at room temperature for 2 h. Aer centrifugation and washing with double distilled water, the prepared AuNPs were dispersed in 1 mL of PBS (0.1 M, pH 7.0) before use.
All the electrochemical experiments were performed in a conventional electrochemical cell containing a three-electrode arrangement at room temperature. CVs were used to characterize the assembly process of the modied electrode in 1 mL of 0.1 M PBS (pH 7.4) containing 5.0 mM[Fe(CN)6]3/4 as the redox probe at a potential range of 0.2 to 0.6 V (vs. SCE) and a scan rate of 100 mV s1. DPV measurements were carried out in 1 mL of 0.1 M PBS (pH 7.0) containing an appropriate amount of glucose at a potential range of 0 to 0.6 V (vs. SCE), a modulation amplitude of 0.05 V, a pulse width of 0.05 s and a sample width of 0.0167 s.
Results and discussion SEM characterization of Fe3O4 and Fe3O4–Au particles Fig. 1 shows the SEM images of Fe3O4 and Fe3O4–Au particles. Clearly, the prepared Fe3O4 particles have a nearly spherical shape and a uniform size with a rough surface morphology (Fig. 1A). The diameter of the Fe3O4 particles is about 250–300 nm. Aer reducing the auric chloride ions with NaBH4 on the surface of the Fe3O4 particles, many ultrathin AuNPs were homogeneously decorated on the surface of the Fe3O4 particles, resulting in the formation of Fe3O4–Au nanocomposites (Fig. 1B). All the above results proved the successful synthesis of the Fe3O4–Au nanocomposites. Electrochemical characterization of the aptasensor
Fabrication of the electrochemical sandwich-type aptasensor The assay protocol for the proposed aptasensor and the corresponding catalysis amplifying principle of the hemin/G-quadruplex conjugated Fe3O4–Au nanocomposites are shown in Scheme 1A. Prior to use, a GCE was rstly polished carefully with 0.3 and 0.05 mm alumina slurries, and then washed ultrasonically in distilled water and ethanol to remove the physically absorbed substance. The pretreated GCE was electrodeposited in 1 mL of 1% HAuCl4 solution at a constant potential of 0.2 V for 30 s to obtain the nano-Au lm modied electrode (depAu/GCE). Following that, 20 mL of NH2–TBA I (2.5 mM) was dropped on the surface of the depAu/GCE and incubated for 16 h at 4 C. Aer washing with PBS buffer, 20 mL of BSA (1%, w/w) was dropped onto the obtained electrode surface and incubated for 40 min at room temperature to block the possible remaining active sites and to eliminate nonspecic absorption. In order to form a sandwich sensing system, 20 mL of TB at different concentrations in Tris–HCl buffer (pH 7.4) was coated onto the above electrode and incubated for 40 min at room temperature. Next, 20 mL of the prepared secondary aptamer was applied to the modied TB/BSA/NH2–TBA I/depAu/GCE and incubated for another 1 h. Aer every step, the obtained electrode was washed
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CVs taken with different modied electrodes in [Fe(CN)6]3/4 solutions are shown in Fig. 2. As can be seen, the bare GCE exhibited a pair of well dened redox peaks (curve a). Upon the electrodeposition of AuNPs on the surface of the bare GCE, the depAu/GCE showed an increased redox current (curve b), which is ascribed to the fact that the AuNPs are a perfect electric conducting material and facilitated the electron transfer. Aer assembly of NH2–TBA I on the electrode surface, the redox current decreased clearly (curve c), indicating that TBA blocked the electron transfer tunnel. When BSA was employed to block the nonspecic sites, an obvious decrease was obtained (curve
Fig. 1 SEM images of Fe3O4 particles (A) and Fe3O4–Au nanocomposites (B).
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Fig. 2 CVs of the bare GCE (a), depAu/GCE (b), NH2–TBA I/depAu/ GCE (c), BSA/NH2–TBA I/depAu/GCE (d), TB/BSA/NH2–TBA I/depAu/ GCE (e) in 1 mL [Fe(CN)6]3/4 (5.0 mM, pH 7.4) at a scan rate of 100 mV s1.
d), which might be due to the fact that BSA as protein biomacromolecules insulated the conductive support. As expected, aer the aptasensor was incubated with 5 nM of thrombin (curve e), the CV response further decreased, owing to the increase of the steric hindrance.
Electrochemical behaviour of the aptasensor and the electron transfer of hemin The signal amplication strategy was accomplished by the corporate catalysis of Fe3O4–Au nanocomposites and hemin/Gquadruplex. In order to demonstrate the feasibility of the proposed pseudobienzyme cascade amplication strategy, the modied electrode incubated with 5 nM thrombin was subjected to DPVs in 1 mL PBS (0.1 M, pH 7.0) under the optimized experimental conditions (see ESI S1†). As can be seen from Fig. 3A, when the aptasensor was incubated with only TB without the secondary aptamer, no electrochemical signal was observed in the scanned potential range (cure a). However, an increased current response appeared aer the sandwich-type reaction of the aptasensor with the secondary aptamer (cure b), suggesting that successful modication of the secondary
Fig. 3 (A) DPV curves of the aptasensor in three different conditions: (a) without secondary aptamer in 1 mL of PBS (0.1 M, pH 7.0), (b) with secondary aptamer in 1 mL of PBS (0.1 M, pH 7.0) and (c) with secondary aptamer in 1 mL of PBS (0.1 M, pH 7.0) containing 40 mM glucose. (B) DPV curves of the aptasensor incubated with two nanocomposites of (a) NH2–TBA II labelled Fe3O4–Au (b) hemin/G-quadruplex conjugated Fe3O4–Au in 1 mL of PBS (0.1 M, pH 7.0) containing 40 mM glucose.
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aptamer had occurred. Upon addition of 40 mM glucose into the detection buffer, an obvious increase of the current response could be observed (curve c). The remarkable enhancement of the signal amplication in our assay protocol for the detection of the target TB could be due to two facts. First, ultrathin AuNPs on the surface of Fe3O4–Au nanocomposites could effectively catalyze the oxidation of glucose to gluconolactone with the production of H2O2 in the presence of dissolved O2. Second, the Fe3O4 cores of the Fe3O4–Au nanocomposites and hemin/ G-quadruplex simultaneously catalyzed the reduction of the obtained H2O2. These results conrmed that the above catalysis strategy promoted the redox reaction of hemin and our proposed amplication strategy was workable. In order to demonstrate the electron transfer of hemin, two different secondary aptamers (NH2–TBA II labelled Fe3O4–Au nanocomposites and hemin/G-quadruplex conjugated Fe3O4– Au nanocomposites) were used. As shown in Fig. 3B, hardly any electrochemical signal was measured when the aptasensor was incubated with NH2–TBA II labelled Fe3O4–Au nanocomposites (curve a). Nevertheless, a clear electrochemical signal was measured aer the aptasensor was incubated with hemin/ G-quadruplex conjugated Fe3O4–Au nanocomposites (curve b), which can be attributed to the electron transfer of hemin. Such results indicated that the observed electrochemical signal of the proposed aptasensor was attributed to the electron transfer of hemin.
Electrochemical comparison of different types of secondary aptamer Fe3O4 particles have the advantages of special magnetic properties and peroxidase-mimicking enzyme activity, acting as cores to immobilize ultrane AuNPs with high GOx-mimicking enzyme activity. When glucose was added into the electrochemical cell, which contained dissolved oxygen, AuNPs could catalytically oxidize glucose and yield H2O2. At the same time, the produced H2O2 was catalyzed by Fe3O4, which makes the articial enzymatic system recycle efficiently. With the large specic surface area and superior conductivity, AuNPs could effectively improve the immobilization of hemin/G-quadruplex components, which could achieve the amplied detection of TB. In order to further investigate the effect of the synthesized Fe3O4–Au nanocomposites on the signal amplication, two types of secondary aptamer were used, such as hemin/G-quadruplex conjugated AuNPs and hemin/G-quadruplex conjugated Fe3O4– Au nanocomposites. As can be seen from Fig. 4, the aptasensor with hemin/G-quadruplex conjugated Au nanocomposites (Fig. 4A) showed a low reduction peak current and electrochemical signal change, indicating a poor conductive performance and low catalytic efficiency to glucose. However, an obviously amplied reduction peak current at the aptasensor with hemin/G-quadruplex conjugated Fe3O4–Au nanocomposites was obtained (Fig. 4B), which can be attributed to the excellent amplifying performance of Fe3O4–Au nanocomposites. Such results indicated the remarkable amplied performance of the proposed secondary aptamer, which may be ascribed to the following reasons: the employment of Fe3O4–Au nanocomposites Analyst, 2014, 139, 1756–1761 | 1759
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shown in Table S1.† The comparison showed that a satisfactory detection limit and wider linear range was obtained using the proposed aptasensor, and this provided powerful evidence of the amplication of the Fe3O4–Au nanocomposites. Selectivity, reproducibility and stability of the proposed aptasensor
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Fig. 4 DPV curves of the different sandwich format aptasensor in the
absence (a) and presence (b) of 40 mM glucose in 1 mL of PBS (0.1 M, pH 7.0) by using different secondary aptamers: (A) hemin/G-quadruplex conjugated Au nanocomposites, and (B) hemin/G-quadruplex conjugated Fe3O4–Au nanocomposites.
with good biocompatibility and intrinsic GOx- and peroxidasemimicking enzyme activity could not only improve the immobilized amount hemin/G-quadruplex, but also enhance the catalysis efficiency of the hemin/G-quadruplex.
Calibration curves obtained for the electrochemical aptasensor The performance of the fabricated aptasensor was evaluated by detecting TB standard solutions in 1 mL of PBS (0.1 M, pH 7.0) containing 40 mM glucose by DPV. As shown in Fig. 5A, the bioelectrocatalytic current intensity of the aptasensor increased with increasing concentrations of TB, as a result of the efficient capture of hemin/G-quadruplex conjugated Fe3O4–Au nanocomposites by the sandwich-type reaction. The DPV signal was linearly dependent on the logarithm of TB concentration in the range of 0.1 pM to 20 nM with a correlation coefficient of R ¼ 0.993 (Fig. 5B). The regression equation was I (mA) ¼ 0.919 log c (nM) 9.028 and a detection limit of 0.013 pM (dened as LOD ¼ 3SB/m, where m is the slope of the corresponding calibration curve and SB is the standard deviation of the blank) was obtained. The above results made it clear that electrochemical amplied TB detection was successfully achieved, and this was attributed to the signicant amplication of the Fe3O4–Au pseudobienzymatic biocatalytic system as expected. Moreover, we also compared this method with other signal amplication strategies for TB detection and the results are
To assess the specicity of the proposed aptasensor for TB detection, control experiments were carried out using other potential interferents, including IgG (50 nM), BSA (50 nM), L-cys (50 nM). As shown in Fig. 6, the presence of the interferents did not show any signicant responses compared with the result from only the target TB (5 nM). Moreover, when IgG, BSA and L-cys were mixed with TB, the DPV signal was not signicantly different to that in the presence of only TB, indicating the good selectivity of the proposed aptasensor for TB detection. To examine the reproducibility, six equally prepared electrodes were incubated with the same concentration of TB (5 nM) under the same conditions. All the electrodes displayed similar electrochemical responses and an RSD of 4.3% was obtained, suggesting the acceptable reproducibility of the aptasensor. The stability of the aptasensor was evaluated by a long-term storage assay. Aer 15 days of storage at 4 C, the aptasensor retained 92.3% of its initial response. The experimental results suggest the acceptable stability and precision of the proposed aptasensor.
Selectivity evaluation of the aptasensor detection of TB (5 nM) against interferents, IgG (50 nM), BSA (50 nM), L-cys (50 nM) and a mixture consisting of the above interferents and TB in 1 mL of PBS (0.1 M, pH 7.0). The error bars indicate the standard deviation of three measurements. Fig. 6
Table 1 Determination of TB in human blood serum (n ¼ 3) by the proposed aptasensora
DPV responses of the aptasensor incubated with different concentrations of TB in 1 mL of PBS (pH 7.0, 0.1 M) containing 40 mM glucose. (B) The calibration plots of DPV peak current versus the logarithm of the TB concentration.
Fig. 5
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Samples
Added TB (nM)
Found TBb (nM)
Recovery (%)
RSD (%)
1 2 3 4 5
0.01 0.1 1.0 5.0 20.0
0.011 0.097 1.05 5.09 18.9
110 97.0 105 102 94.5
5.6 6.4 3.9 7.3 6.2
a
The experimental measurements were accomplished by the DPV in 1 mL PBS (0.1 M, pH 7.0) at room temperature containing 40 mM glucose. b The values were the average values from three measurements.
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Real sample analysis of TB
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According to a previous report, no TB exists in healthy human serum. Therefore, we investigated the practical applicability of the aptasensor by spiking the target TB at different concentrations into 10-fold-diluted serum samples for the preparation of several samples. Then, the obtained samples were surveyed by the proposed aptasensor and the results are shown in Table 1. Recovery and relative standard deviation values obtained ranged from 110% to 94.5% and 3.9% to 7.3%, respectively, indicating the excellent potential of the aptasensor for analytical applications in protein detection.
Conclusion In conclusion, in the present study a sandwich-type electrochemical aptasensor was successfully fabricated for the detection of TB with the use of Fe3O4–Au nanocomposites and hemin/G-quadruplex for signal amplication. On the one hand, Fe3O4–Au nanocomposites as nanocarriers exhibited good biocompatibility and GOx- and peroxidase-mimicking enzyme activities, which could catalyze the oxidization of glucose and simultaneously produce H2O2 with high local concentrations and low transfer loss. On the other hand, the hemin/G-quadruplex simultaneously acted as an HRP-mimicking DNAzyme and electrochemical signal label, thus considerably enhancing the sensitivity of the proposed aptasensor. Moreover, the present aptasensor performed well in the detection of TB with a wide linear range, low detection limit and good selectivity. In view of these advantages, we anticipate that this method can be readily expanded for clinical applications.
Acknowledgements This research was supported by the National Natural Science Foundation (NNSF) of China (21075100), the State Key Laboratory of Electroanalytical Chemistry (SKLEAC2010009), and the High Technology Project Foundation of Southwest University (XSGX02).
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An amplified electrochemical aptasensor for thrombin detection based on pseudobienzymic Fe3O4–Au nanocomposites and electroactive hemin/G-quadruplex as signal enhancers† Pei Jing, Wenju Xu,* Huayu Yi, Yongmei Wu, Lijuan Bai and Ruo Yuan* A sensitive and selective electrochemical aptasensor for thrombin detection was constructed based on hemin/G-quadruplex as the signal label and Fe3O4–Au nanocomposites with glucose oxidase (GOx-) and peroxide-mimicking enzyme activity as the signal enhancers. Due to their large surface area and good biocompatibility, Fe3O4–Au nanocomposites were employed to immobilize electroactive hemin/Gquadruplex, which was formed by the conjugation between a single-stranded guanine-rich nucleic acid and hemin. Based on the GOx-mimicking enzyme activity, Au nanoparticles on the surface of the Fe3O4–Au nanocomposites effectively catalyzed the oxidization of glucose in the presence of dissolved O2, accompanied by the production of H2O2. Both the Fe3O4 cores of Fe3O4–Au nanocomposites and hemin/G-quadruplex with H2O2-mimicking enzyme activity could catalyze the reduction of the
Received 3rd December 2013 Accepted 3rd January 2014
generated H2O2, which promoted the electron transfer of hemin and amplified the electrochemical signal. The proposed electrochemical aptasensor had a wide dynamic linear range of 0.1 pM to 20 nM
DOI: 10.1039/c3an02237d
with a lower detection limit of 0.013 pM, which provided a promising method for a sensitive assay for
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the detection of proteins in electrochemical aptasensors.
Introduction Thrombin (TB), is a kind of serine protease of the blood stream chymotrypsin family, it plays an essential role in the production of insoluble brin through the proteolytic cleavage of soluble brinogen.1 In addition, it is involved in some physiological and pathological processes, including leukemia, arterial thrombosis and liver disease.2 Therefore, highly sensitive and cost-efficient methods for the quantitative detection of TB in clinical diagnosis and treatment are crucial. In recent years, great effort has been made to design aptamer-based assays, such as uorescence,3 chemiluminescence,4 surface plasmon resonance,5 colorimetry,6 quartz crystal microbalance7 and electrochemistry,8–10 for TB detection. Compared with other methods that suffer from the disadvantages of complex operation, large-scale and high-cost instrumentation, the aptamer-based electrochemical method has merits of fast response, high sensitivity, portability, simplicity and inexpensive apparatus,11 and has been widely used in the sensitive detection of TB. In these electrochemical aptasensors, many redox mediator labels, such as methylene blue,12 thionine,13 toluidine blue,14 ferrocene15 and so on, have been used to generate an Education Ministry Key Laboratory on Luminescence and Real-TimeAnalysis, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, People's Republic of China. E-mail: [email protected] † Electronic supplementary 10.1039/c3an02237d
information
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(ESI)
available.
See
DOI:
electrochemical signal. Alternatively, hemin [Fe(III)-protoporphyrin IX], the active cofactor for catalases,16 can also generate an electrochemical signal,17 which makes it possible for hemin to act as an excellent electroactive material for constructing an aptasensor. In addition, hemin can intercalate into a singlestranded guanine-rich nucleic acid with a unique G-quadruplex structure to form a hemin/G-quadruplex structure with hydrogen peroxide (HRP)-mimicking DNAzyme activity.18,19 The hemin/ G-quadruplex biocatalytic structure, which possesses the advantages of inexpensive production, greater stability against hydrolysis, relatively easy labelling and which can greatly enhance the response signal,20,21 has been used to detect metal ions,22,23 proteins,24 DNAs25 and small molecules.26 Recently, electroactive or photoactive molecular labelled nanomaterials have been popularly employed in such aptamerbased detection systems to realize great signal amplication and improve the sensitivity of the method.27–30 Among them, Fe3O4 magnetic nanoparticles (Fe3O4 MNPs) with a unique superparamagnetic activity have been widely exploited in biology and medicine to detect small molecules,31 and to separate and detect proteins.32 In previous research, Fe3O4 MNPs have been generally considered to be biology inactive. However, Wei and coworkers have discovered that Fe3O4 MNPs could exhibit intrinsic peroxidase-mimicking enzyme activity.33 Based on these merits, graphene oxide–Fe3O4 magnetic nanocomposites were employed to detect glucose by using the peroxidase-mimicking enzyme activity
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of Fe3O4 MNPs.34 Lately, He et al. prepared discrete and monodispersed Fe3O4–Au@mesoporous SiO2 (MS) microspheres with high GOx- and peroxidase-mimicking enzyme activities.35 The ultrane (4.2–7.2 nm) Au nanoparticles (AuNPs) on the surface of these nanocomposites, acting as a GOx-mimicking enzyme, can catalyze glucose to gluconolactone coupled with the generation of H2O2 in the presence of the dissolved O2. Then, Fe3O4 acts as a peroxidase-mimicking enzyme and further catalyzes the reduction of the obtained H2O2. Owing to its remarkable pseudobienzyme activity, this kind of nanocomposite could be used to prepare a signal amplied aptasensor for the detection of TB. In this work, we designed a sandwich-type electrochemical aptasensor by utilizing pseudobienzymic Fe3O4–Au nanocomposites and an electroactive hemin/G-quadruplex structure as signal enhancers for the sensitive detection of TB. Fe3O4–Au nanocomposites with a large surface area and pseudobienzyme activity were employed for the immobilization of hemin/Gquadruplex to form hemin/G-quadruplex conjugated Fe3O4–Au nanocomposites (secondary aptamer). The target protein was sandwiched between the primary amido-terminated thrombin aptamer I (NH2–TBA I) and the prepared secondary aptamer. In the presence of glucose, active AuNPs of Fe3O4–Au nanocomposites could oxidize glucose and simultaneously produce H2O2 in the presence of dissolved O2. Furthermore, the Fe3O4 cores of Fe3O4–Au nanocomposites and hemin/G-quadruplex could cooperative to electrocatalyze the reduction of H2O2 and to promote the electron transport of hemin, in order to achieve a signicantly amplied electrochemical signal. The results indicated an effective way to quantitatively detect the target TB, according to the linearity between the logarithm of TB concentration and the current response. This amplied strategy could be applied in the development of a simple and sensitive analytical method for the detection of proteins.
Experimental Chemicals and materials TB, hemin, L-cysteine (L-cys), gold chloride (HAuCl4), bovine serum albumin (BSA) and human IgG were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Trisodium citrate, NaBH4, sodium acetate (NaAc), FeCl3$6H2O and ethylene glycol were obtained from Kelong Chemical Company (Chengdu, China). Glucose was obtained from Sinopharm Chemical Reagent Co. Ltd. (China). The human serum samples were obtained from the Ninth People's Hospital of Chongqing (China). Tris-hydroxymethylaminomethane hydrochloride (Tris) was provided by Roche (Switzerland). Thrombin aptamer (TBA): 50 –NH2–(CH2)6–GGTTGGTGTGGTTGG-30 was purchased from Sangon BiotechCo., Ltd (Shanghai, China). Phosphate buffered solution (PBS) with different pH was prepared using 0.1 M Na2HPO4, 0.1 M KH2PO4 and 0.1 M KCl and served as the working buffer throughout the experiment. Tris–HCl buffer (20 mM, pH 7.4) was prepared with 140 mM NaCl, 5 mM KCl, 1 mM CaCl2 and 1 mM MgCl2, and was used as the aptamer buffer. The prepared solutions were kept at 4 C before use. All the other chemicals used were of analytical grade
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and were used directly as received. Double distilled water was used throughout this work. Apparatus Differential pulse voltammetry measurements (DPVs) and cyclic voltammetry measurements (CVs) were performed with a CHI 660D electrochemical workstation (Shanghai Chenhua Instrument, China). A three electrode system, including a modied glassy carbon electrode (GCE, F ¼ 4 mm) as the working electrode, a platinum wire as the counter electrode, a saturated calomel electrode (SCE) as the reference electrode, was employed for the above electrochemical measurements. The scanning electron micrographs (SEM) were taken with a scanning electron microscope (SEM, S-4800, Hitachi, Tokyo, Japan). Synthesis of Fe3O4–Au nanocomposites Fe3O4–Au nanocomposites were prepared via a hydrothermal method according to the literature with minor revision.36 In brief, 0.70 g of FeCl3$6H2O, 0.28 g of trisodium citrate were rst dispersed in 20 mL of ethylene glycol, aer that 1.2 g of NaAc was added into the resulting solution under continual stirring until the mixture became homogeneous and the colour was yellow. Subsequently, the obtained solution was maintained at 200 C for 10 h in a Teon-lined stainless-steel autoclave. When the autoclave had cooled to room temperature, the resulting precipitate was collected by an external magnetic eld. The black products were washed several times with ethanol and nitrogen-saturated water alternately to remove most of the matrices, including trisodium citrate and ethylene glycol. Finally the particles were dried at 60 C in a vacuum drying oven. The prepared Fe3O4 particles (20 mg) were added to 25 mL of nitrogen-saturated water with sonication for 5 min. Aerwards, 405 mL of HAuCl4 (1%) was added to the above dispersion and magnetically stirred constantly for 30 min. 1 mL of freshly prepared NaBH4 (0.15 M) was added into the above solution under vigorous stirring, and then the mixture was stirred for another 30 min. The resulting product was separated and washed with nitrogen-saturated water several times using a magnet. Lastly, Fe3O4–Au nanocomposites were suspended in 1 mL of PBS (0.1 M, pH 7.0) before use. Synthesis of hemin/G-quadruplex conjugated Fe3O4–Au nanocomposites (secondary aptamer) Based on the conjugation of AuNPs to TBA via the reaction with the amine groups of the aptamer, the secondary aptamer was prepared as follows (Scheme 1B): rstly, 60 mL of NH2–TBA II (100 nM) was injected into the prepared solution and gently stirred for 12 h at 4 C. Secondly, 0.5 mg of hemin was added into the above solution and stirred for 40 min. At this time, the hemin/G-quadruplex biocatalyst structure was obtained. Finally, 200 mL of BSA (w/w, 1%) aqueous solution was added into the obtained mixture and stirred for another 40 min to block the unoccupied active sites of the Fe3O4–Au nanocomposites. Aer centrifugation and washing, the hemin/ G-quadruplex conjugated Fe3O4–Au nanocomposites was Analyst, 2014, 139, 1756–1761 | 1757
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with distilled water to remove physically absorbed species. DPV curves of the proposed aptasensor aer incubation with the secondary aptamer in the absence (I0) and presence (I) of 40 mM glucose in 1 mL of PBS (0.1 M, pH 7.0) are shown in Scheme 1C.
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Experimental measurements
Scheme 1 (A) Schematic illustration of the stepwise fabrication of the electrochemical aptasensor. (B) Procedure for the preparation of hemin/G-quadruplex conjugated Fe3O4–Au nanocomposites (secondary aptamer). (C) DPV cures of the proposed aptasensor after incubation with the secondary aptamer in the absence (I0) and presence (I) of 40 mM glucose in 1 mL of PBS (0.1 M, pH 7.0).
resuspended in 1 mL of Tris–HCl buffer and stored at 4 C for further use. In order to study the amplication properties of the Fe3O4– Au nanocomposites, hemin/G-quadruplex conjugated Au nanocomposites were also prepared in a similar way but without Fe3O4. AuNPs (5 nm) were prepared according to the reported method with some modications by using KBH4 as a reductant and sodium citrate as a stabilizer.36 Briey, 1 mL of HAuCl4 (1%) and 2 mL of sodium citrate (0.03 M) were added to 50 mL of double distilled water and stirred, followed by the addition of 1 mL of freshly prepared NaBH4 (0.1 M). The resulting solution whose colour changed to wine red was maintained at room temperature for 2 h. Aer centrifugation and washing with double distilled water, the prepared AuNPs were dispersed in 1 mL of PBS (0.1 M, pH 7.0) before use.
All the electrochemical experiments were performed in a conventional electrochemical cell containing a three-electrode arrangement at room temperature. CVs were used to characterize the assembly process of the modied electrode in 1 mL of 0.1 M PBS (pH 7.4) containing 5.0 mM[Fe(CN)6]3/4 as the redox probe at a potential range of 0.2 to 0.6 V (vs. SCE) and a scan rate of 100 mV s1. DPV measurements were carried out in 1 mL of 0.1 M PBS (pH 7.0) containing an appropriate amount of glucose at a potential range of 0 to 0.6 V (vs. SCE), a modulation amplitude of 0.05 V, a pulse width of 0.05 s and a sample width of 0.0167 s.
Results and discussion SEM characterization of Fe3O4 and Fe3O4–Au particles Fig. 1 shows the SEM images of Fe3O4 and Fe3O4–Au particles. Clearly, the prepared Fe3O4 particles have a nearly spherical shape and a uniform size with a rough surface morphology (Fig. 1A). The diameter of the Fe3O4 particles is about 250–300 nm. Aer reducing the auric chloride ions with NaBH4 on the surface of the Fe3O4 particles, many ultrathin AuNPs were homogeneously decorated on the surface of the Fe3O4 particles, resulting in the formation of Fe3O4–Au nanocomposites (Fig. 1B). All the above results proved the successful synthesis of the Fe3O4–Au nanocomposites. Electrochemical characterization of the aptasensor
Fabrication of the electrochemical sandwich-type aptasensor The assay protocol for the proposed aptasensor and the corresponding catalysis amplifying principle of the hemin/G-quadruplex conjugated Fe3O4–Au nanocomposites are shown in Scheme 1A. Prior to use, a GCE was rstly polished carefully with 0.3 and 0.05 mm alumina slurries, and then washed ultrasonically in distilled water and ethanol to remove the physically absorbed substance. The pretreated GCE was electrodeposited in 1 mL of 1% HAuCl4 solution at a constant potential of 0.2 V for 30 s to obtain the nano-Au lm modied electrode (depAu/GCE). Following that, 20 mL of NH2–TBA I (2.5 mM) was dropped on the surface of the depAu/GCE and incubated for 16 h at 4 C. Aer washing with PBS buffer, 20 mL of BSA (1%, w/w) was dropped onto the obtained electrode surface and incubated for 40 min at room temperature to block the possible remaining active sites and to eliminate nonspecic absorption. In order to form a sandwich sensing system, 20 mL of TB at different concentrations in Tris–HCl buffer (pH 7.4) was coated onto the above electrode and incubated for 40 min at room temperature. Next, 20 mL of the prepared secondary aptamer was applied to the modied TB/BSA/NH2–TBA I/depAu/GCE and incubated for another 1 h. Aer every step, the obtained electrode was washed
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CVs taken with different modied electrodes in [Fe(CN)6]3/4 solutions are shown in Fig. 2. As can be seen, the bare GCE exhibited a pair of well dened redox peaks (curve a). Upon the electrodeposition of AuNPs on the surface of the bare GCE, the depAu/GCE showed an increased redox current (curve b), which is ascribed to the fact that the AuNPs are a perfect electric conducting material and facilitated the electron transfer. Aer assembly of NH2–TBA I on the electrode surface, the redox current decreased clearly (curve c), indicating that TBA blocked the electron transfer tunnel. When BSA was employed to block the nonspecic sites, an obvious decrease was obtained (curve
Fig. 1 SEM images of Fe3O4 particles (A) and Fe3O4–Au nanocomposites (B).
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Fig. 2 CVs of the bare GCE (a), depAu/GCE (b), NH2–TBA I/depAu/ GCE (c), BSA/NH2–TBA I/depAu/GCE (d), TB/BSA/NH2–TBA I/depAu/ GCE (e) in 1 mL [Fe(CN)6]3/4 (5.0 mM, pH 7.4) at a scan rate of 100 mV s1.
d), which might be due to the fact that BSA as protein biomacromolecules insulated the conductive support. As expected, aer the aptasensor was incubated with 5 nM of thrombin (curve e), the CV response further decreased, owing to the increase of the steric hindrance.
Electrochemical behaviour of the aptasensor and the electron transfer of hemin The signal amplication strategy was accomplished by the corporate catalysis of Fe3O4–Au nanocomposites and hemin/Gquadruplex. In order to demonstrate the feasibility of the proposed pseudobienzyme cascade amplication strategy, the modied electrode incubated with 5 nM thrombin was subjected to DPVs in 1 mL PBS (0.1 M, pH 7.0) under the optimized experimental conditions (see ESI S1†). As can be seen from Fig. 3A, when the aptasensor was incubated with only TB without the secondary aptamer, no electrochemical signal was observed in the scanned potential range (cure a). However, an increased current response appeared aer the sandwich-type reaction of the aptasensor with the secondary aptamer (cure b), suggesting that successful modication of the secondary
Fig. 3 (A) DPV curves of the aptasensor in three different conditions: (a) without secondary aptamer in 1 mL of PBS (0.1 M, pH 7.0), (b) with secondary aptamer in 1 mL of PBS (0.1 M, pH 7.0) and (c) with secondary aptamer in 1 mL of PBS (0.1 M, pH 7.0) containing 40 mM glucose. (B) DPV curves of the aptasensor incubated with two nanocomposites of (a) NH2–TBA II labelled Fe3O4–Au (b) hemin/G-quadruplex conjugated Fe3O4–Au in 1 mL of PBS (0.1 M, pH 7.0) containing 40 mM glucose.
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aptamer had occurred. Upon addition of 40 mM glucose into the detection buffer, an obvious increase of the current response could be observed (curve c). The remarkable enhancement of the signal amplication in our assay protocol for the detection of the target TB could be due to two facts. First, ultrathin AuNPs on the surface of Fe3O4–Au nanocomposites could effectively catalyze the oxidation of glucose to gluconolactone with the production of H2O2 in the presence of dissolved O2. Second, the Fe3O4 cores of the Fe3O4–Au nanocomposites and hemin/ G-quadruplex simultaneously catalyzed the reduction of the obtained H2O2. These results conrmed that the above catalysis strategy promoted the redox reaction of hemin and our proposed amplication strategy was workable. In order to demonstrate the electron transfer of hemin, two different secondary aptamers (NH2–TBA II labelled Fe3O4–Au nanocomposites and hemin/G-quadruplex conjugated Fe3O4– Au nanocomposites) were used. As shown in Fig. 3B, hardly any electrochemical signal was measured when the aptasensor was incubated with NH2–TBA II labelled Fe3O4–Au nanocomposites (curve a). Nevertheless, a clear electrochemical signal was measured aer the aptasensor was incubated with hemin/ G-quadruplex conjugated Fe3O4–Au nanocomposites (curve b), which can be attributed to the electron transfer of hemin. Such results indicated that the observed electrochemical signal of the proposed aptasensor was attributed to the electron transfer of hemin.
Electrochemical comparison of different types of secondary aptamer Fe3O4 particles have the advantages of special magnetic properties and peroxidase-mimicking enzyme activity, acting as cores to immobilize ultrane AuNPs with high GOx-mimicking enzyme activity. When glucose was added into the electrochemical cell, which contained dissolved oxygen, AuNPs could catalytically oxidize glucose and yield H2O2. At the same time, the produced H2O2 was catalyzed by Fe3O4, which makes the articial enzymatic system recycle efficiently. With the large specic surface area and superior conductivity, AuNPs could effectively improve the immobilization of hemin/G-quadruplex components, which could achieve the amplied detection of TB. In order to further investigate the effect of the synthesized Fe3O4–Au nanocomposites on the signal amplication, two types of secondary aptamer were used, such as hemin/G-quadruplex conjugated AuNPs and hemin/G-quadruplex conjugated Fe3O4– Au nanocomposites. As can be seen from Fig. 4, the aptasensor with hemin/G-quadruplex conjugated Au nanocomposites (Fig. 4A) showed a low reduction peak current and electrochemical signal change, indicating a poor conductive performance and low catalytic efficiency to glucose. However, an obviously amplied reduction peak current at the aptasensor with hemin/G-quadruplex conjugated Fe3O4–Au nanocomposites was obtained (Fig. 4B), which can be attributed to the excellent amplifying performance of Fe3O4–Au nanocomposites. Such results indicated the remarkable amplied performance of the proposed secondary aptamer, which may be ascribed to the following reasons: the employment of Fe3O4–Au nanocomposites Analyst, 2014, 139, 1756–1761 | 1759
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shown in Table S1.† The comparison showed that a satisfactory detection limit and wider linear range was obtained using the proposed aptasensor, and this provided powerful evidence of the amplication of the Fe3O4–Au nanocomposites. Selectivity, reproducibility and stability of the proposed aptasensor
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Fig. 4 DPV curves of the different sandwich format aptasensor in the
absence (a) and presence (b) of 40 mM glucose in 1 mL of PBS (0.1 M, pH 7.0) by using different secondary aptamers: (A) hemin/G-quadruplex conjugated Au nanocomposites, and (B) hemin/G-quadruplex conjugated Fe3O4–Au nanocomposites.
with good biocompatibility and intrinsic GOx- and peroxidasemimicking enzyme activity could not only improve the immobilized amount hemin/G-quadruplex, but also enhance the catalysis efficiency of the hemin/G-quadruplex.
Calibration curves obtained for the electrochemical aptasensor The performance of the fabricated aptasensor was evaluated by detecting TB standard solutions in 1 mL of PBS (0.1 M, pH 7.0) containing 40 mM glucose by DPV. As shown in Fig. 5A, the bioelectrocatalytic current intensity of the aptasensor increased with increasing concentrations of TB, as a result of the efficient capture of hemin/G-quadruplex conjugated Fe3O4–Au nanocomposites by the sandwich-type reaction. The DPV signal was linearly dependent on the logarithm of TB concentration in the range of 0.1 pM to 20 nM with a correlation coefficient of R ¼ 0.993 (Fig. 5B). The regression equation was I (mA) ¼ 0.919 log c (nM) 9.028 and a detection limit of 0.013 pM (dened as LOD ¼ 3SB/m, where m is the slope of the corresponding calibration curve and SB is the standard deviation of the blank) was obtained. The above results made it clear that electrochemical amplied TB detection was successfully achieved, and this was attributed to the signicant amplication of the Fe3O4–Au pseudobienzymatic biocatalytic system as expected. Moreover, we also compared this method with other signal amplication strategies for TB detection and the results are
To assess the specicity of the proposed aptasensor for TB detection, control experiments were carried out using other potential interferents, including IgG (50 nM), BSA (50 nM), L-cys (50 nM). As shown in Fig. 6, the presence of the interferents did not show any signicant responses compared with the result from only the target TB (5 nM). Moreover, when IgG, BSA and L-cys were mixed with TB, the DPV signal was not signicantly different to that in the presence of only TB, indicating the good selectivity of the proposed aptasensor for TB detection. To examine the reproducibility, six equally prepared electrodes were incubated with the same concentration of TB (5 nM) under the same conditions. All the electrodes displayed similar electrochemical responses and an RSD of 4.3% was obtained, suggesting the acceptable reproducibility of the aptasensor. The stability of the aptasensor was evaluated by a long-term storage assay. Aer 15 days of storage at 4 C, the aptasensor retained 92.3% of its initial response. The experimental results suggest the acceptable stability and precision of the proposed aptasensor.
Selectivity evaluation of the aptasensor detection of TB (5 nM) against interferents, IgG (50 nM), BSA (50 nM), L-cys (50 nM) and a mixture consisting of the above interferents and TB in 1 mL of PBS (0.1 M, pH 7.0). The error bars indicate the standard deviation of three measurements. Fig. 6
Table 1 Determination of TB in human blood serum (n ¼ 3) by the proposed aptasensora
DPV responses of the aptasensor incubated with different concentrations of TB in 1 mL of PBS (pH 7.0, 0.1 M) containing 40 mM glucose. (B) The calibration plots of DPV peak current versus the logarithm of the TB concentration.
Fig. 5
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Added TB (nM)
Found TBb (nM)
Recovery (%)
RSD (%)
1 2 3 4 5
0.01 0.1 1.0 5.0 20.0
0.011 0.097 1.05 5.09 18.9
110 97.0 105 102 94.5
5.6 6.4 3.9 7.3 6.2
a
The experimental measurements were accomplished by the DPV in 1 mL PBS (0.1 M, pH 7.0) at room temperature containing 40 mM glucose. b The values were the average values from three measurements.
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Real sample analysis of TB
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According to a previous report, no TB exists in healthy human serum. Therefore, we investigated the practical applicability of the aptasensor by spiking the target TB at different concentrations into 10-fold-diluted serum samples for the preparation of several samples. Then, the obtained samples were surveyed by the proposed aptasensor and the results are shown in Table 1. Recovery and relative standard deviation values obtained ranged from 110% to 94.5% and 3.9% to 7.3%, respectively, indicating the excellent potential of the aptasensor for analytical applications in protein detection.
Conclusion In conclusion, in the present study a sandwich-type electrochemical aptasensor was successfully fabricated for the detection of TB with the use of Fe3O4–Au nanocomposites and hemin/G-quadruplex for signal amplication. On the one hand, Fe3O4–Au nanocomposites as nanocarriers exhibited good biocompatibility and GOx- and peroxidase-mimicking enzyme activities, which could catalyze the oxidization of glucose and simultaneously produce H2O2 with high local concentrations and low transfer loss. On the other hand, the hemin/G-quadruplex simultaneously acted as an HRP-mimicking DNAzyme and electrochemical signal label, thus considerably enhancing the sensitivity of the proposed aptasensor. Moreover, the present aptasensor performed well in the detection of TB with a wide linear range, low detection limit and good selectivity. In view of these advantages, we anticipate that this method can be readily expanded for clinical applications.
Acknowledgements This research was supported by the National Natural Science Foundation (NNSF) of China (21075100), the State Key Laboratory of Electroanalytical Chemistry (SKLEAC2010009), and the High Technology Project Foundation of Southwest University (XSGX02).
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