Biosensors and Bioelectronics 54 (2014) 415–420
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A pseudo triple-enzyme cascade amplified aptasensor for thrombin detection based on hemin/G-quadruplex as signal label Huayu Yi, Wenju Xu n, Yali Yuan, Lijuan Bai, Yongmei Wu, Yaqin Chai, Ruo Yuan n Key Laboratory on Luminescence and Real-Time Analysis, School of Chemistry and Chemical Engineering, Southwest University, The Key Laboratory of Eco-environments in Three Gorges Reservoir Region, Chongqing 400715, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 25 July 2013 Received in revised form 7 November 2013 Accepted 7 November 2013 Available online 18 November 2013
In this work, a pseudo triple-enzyme cascade amplified electrochemical aptasensor based on hemin/Gquadruplex as signal label for thrombin (TB) was constructed and the amplified electrochemical signal was achieved by the corporate catalysis of alcohol dehydrogenase-graphene sheets (ADH-GSs) bionanocomposite and hemin/G-quadruplex, which simultaneously acted as NADH oxidase and HRP-mimicking DNAzyme. Through “sandwich” reaction, hemin/G-quadruplex labeled gold nanoparticles-ADH-GSs bionanocomposite (AuNPs-ADH-GSs) was captured on electrode surface and thus obtained the electrochemical signal. After the addition of ethanol into the electrolytic cell, ADH availably catalyzed the oxidation of ethanol with the reduction of NAD þ to NADH. Then, hemin/G-quadruplex as NADH oxidase catalyzed the oxidization of NADH, accompanying with the production of H2O2. Simultaneously, hemin/Gquadruplex as HRP-mimicking DNAzyme catalyzed the reduction of the generated H2O2. Such a catalysis strategy greatly promoted the electron transfer of hemin and resulted in the specific enhancement of electrochemical signal. The proposed TB aptasensor achieved a linear range of 1 pM–50 nM with a detection limit of 0.3 pM (defined as S/N¼3). In addition, it showed satisfying stability and reproducibility, good specificity and sensitivity, indicating promising application for the detection of various proteins in clinical analysis. & 2013 Elsevier B.V. All rights reserved.
Keywords: Pseudo triple-enzyme Aptasensor Thrombin Alcohol dehydrogenase-graphene Hemin/G-quadruplex
1. Introduction Electrochemical aptamer-based sensors (aptasensors), based on the binding-induced conformational changes of redox-tagged and surface-confined aptamers (Zuo et al., 2007), have played an important role in the detection of proteins and small molecules (Wu et al., 2012; Cheng et al., 2012; Du et al., 2009), owing to the superior advantages, such as simple instrumentation, low cost, fast response, portability and high sensitivity (Liu et al., 2012; Chen et al., 2012). The aptasensors for this usage often involve in some redox mediators labeling, such as ferrocene (Liu et al., 2010), methylene blue (Fu et al., 2011), thionine (Tang et al., 2012) and so on. Alternatively, hemin referred to iron-containing porphyrin is the active center of hemoglobin (Kaneko et al., 2013; Cao et al., 2012) and can also offer electrochemical signal, which provides its possibility for constructing aptasensor. On the basis of this, an electrochemical thrombin (TB) aptasensor based on the electron transfer of hemin has been reported with a detection limit of 0.1 nM (Jiang et al., 2013). Besides, hemin also possesses excellent peroxidase activity when it forms the hemin/G-quadruplex structure via intercalating hemin into a guanine-rich single-stranded
n
Corresponding authors. Tel./fax: þ86 23 68252277. E-mail address: [email protected] (W. Xu).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.11.036
DNA with unique G-quadruplex structure (Meng et al., 2013). With advantages of good stability, inexpensive production and facile self-replication (Zhang et al., 2011; Chen et al., 2009), hemin/Gquadruplex structure has been used as peroxidase mimetic for detecting proteins (Li et al., 2008), metal ions (Li et al., 2010), small molecules (Li et al., 2007), and DNAs (Xiao et al., 2004). In addition, Golub and coworkers have proved that the hemin/ G-quadruplex structure can simultaneously act as horseradish peroxidase (HRP)-mimicking DNAzyme and NADH oxidase (Golub et al., 2011). Inspired by this property, an amplified TB electrochemical aptasensor was successfully fabricated by using the pseudobienzyme ability of hemin/G-quadruplex in our previous work (Yuan et al., 2012). Nevertheless, the combination of hemin/G-quadruplex as signal label with its remarkable catalytic performance has been received little attention in the field of electrochemical aptasensor. In recent years, multiple nanomaterials with unique electrical, physical and chemical properties have emerged for fabricating signal amplified biosensors (Guo et al., 2011; Li et al., 2012; He et al., 2011). Among them, graphene is widely used in biosensor applications with merits of flexibility, large surface area, excellent electrical conductivities and good mechanical properties (Chen et al., 2011; Lee et al., 2010). Thus, it is capable for graphene to immobilize a large amount of nanoparticles or enzymes (Zhang et al., 2011; Zeng et al., 2010), providing a promising approach for
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signal amplification in electrical biosensing (Bai et al., 2012). Additionally, the features of graphene endow the ability for facilitating electron transfer of redox mediators, which paves the way for fabrication of sensitive electrochemical aptasensors. With above advantages, graphene was employed as an enzyme immobilized platform in this work. Firstly, we prepared an alcohol dehydrogenase-graphene sheets (ADH-GSs) bionanocomposite, followed by assembly of plentiful gold nanoparticles (AuNPs) through the interaction between AuNPs and NH2 groups in ADH. In this way, a large amount of ADH and AuNPs could be immobilized on the graphene sheet with large surface area. Moreover, AuNPs could improve the conductivity of ADH-GSs bionanocomposite and further act as the platform for immobilizing thrombin aptamer (TBA). The obtained AuNPs-ADH-GSs bionanocomposite was then labled with hemin/G-quadruplex formed by intercalating hemin into the amino-terminated TBA. Finally, the hemin/G-quadruplex labeled AuNPs-ADH-GSs bionanocomposite (secondary thrombin aptamer: TBA II) with good redox and electrocatalytic ability was formed. Through “sandwich” reaction, TBA II was captured on the electrode surface, resulting in an electrochemical signal. Therefore, a pseudo triple-enzyme cascade amplified aptasensor was successfully designed for TB detection based on the corporate catalysis of ADH-GSs bionanocomposite and hemin/G-quadruplex, which simultaneously acted as NADH oxidase and HRP-mimicking DNAzyme. In the presence of ethanol and NAD þ , ADH effectively catalyzed the oxidation of ethanol to acetaldehyde, coupling with the production of NADH. Then, hemin/G-quadruplex acted as NADH oxidase, assisting the oxidation of NADH accompanying with the generation of H2O2 in the presence of dissolved O2. As HRPmimicking DNAzyme, hemin/G-quadruplex simultaneously catalyzed the reduction of H2O2. Finally, the electrochemical signal was amplified. The proposed aptasensor showed good sensitivity for quantitative determination of TB, which indicated a promising strategy for sensitive, simple detection of proteins in clinical applications.
2. Experimental 2.1. Materials and reagents Hemin, alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae, β-nicotinamide adenine dinucleotide hydrate (NAD þ ), thrombin (TB), bovine serum albumin (BSA), human IgG, hemoglobin (Hb), gold chloride (HAuCl4) were obtained from Sigma-Aldrich Chem. Co. (St. Louis, MO, USA). Graphene oxide (GO) was obtained from Nanjing XianFeng Nano Co. (Nanjing, China). Poly(diallyldimethylammonium chloride) (PDDA) was obtained from Beijing Chemical Reagent Co. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was provided by Shanghai Medpep Co. (Shanghai, China). L-cysteine (L-cys) was purchased from Kangan Amino Acid Company (Shanghai, China). Tris-hydroxymethylaminomethane hydrochloride (Tris) was purchased from Roche (Switzerland). Thrombin aptamer (TBA): 5′-NH2-(CH2)6-GGTTGGTGTGGTTGG-3′ was purchased from Sangon Biotech (Shanghai) Co., Ltd. 0.1 M PBS (pH 7.0) containing 10 mM Na2HPO4, 10 mM NaH2PO4 and 2 mM MgCl2 was used as working buffer solution, 20 mM Tris–HCl buffer (pH 7.4) containing 140 mM NaCl, 5 mM KCl, 1 mM CaCl2 and 1 mM MgCl2 was used as binding buffer solution. All other chemicals were of reagent grade and used as received. Double distilled water was used throughout the study. 2.2. Apparatus and measurements Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed with a CHI 660D electrochemical workstation (Shanghai Chenhua instrument, China). The scanning
electron micrographs were taken with scanning electron microscope (SEM, S-4800, Hitachi). All electrochemical experiments were conducted on a three-electrode system with a platinum wire as auxiliary electrode, a saturated calomel as reference electrode (SCE) and a bare or modified glassy carbon electrode (GCE, Ф ¼4 mm) as the working electrode. CV experiments were performed in 1.0 mL (5 mM) of [Fe(CN)6]3 /4 solution with the potential ranging from 0.2 V to 0.6 V. DPV measurements were carried out in 1.0 mL of 0.1 M PBS (pH 7.0) containing 80 μL NAD þ (0.25 mM) and 80 μL absolute ethanol. The scan rate was 100 mV/ s. The DPV measurements were taken with 4 mV of potential incremental, 50 mV of amplitude, 50 s of pulse width, and 0.0167 s of sample width with the potential ranging from 0.6 V to 0 V. 2.3. Preparation of ADH-graphene sheets (ADH-GSs) bionanocomposite Firstly, the PDDA-protected GSs with positive charge were synthesized according to the literature (Li et al., 2011) with a slight modification: A stable dispersion of exfoliated GO sheets (50 mL, 1 mg/mL) was mixed with PDDA (0.2 mL, 20%) and stirred for 30 min. Then, 0.5 mL hydrazine hydrate (80%) was added into the mixture with stirring for 24 h at 90 1C. Subsequently, the obtained black reaction product was centrifuged, washed with double distilled water and redispersed in water under mild sonication. The obtained PDDA-protected GSs were investigated by SEM observation, which is shown in Fig. S1A (see Supplementary materials S1). The ADH-GSs bionanocomposite was prepared as follows. Briefly, 0.5 mL PDDA-protected GSs prepared before was mixed with 0.5 mL ADH (pI ¼5.4) solution (2 mg/mL) prepared in PBS solution (pH 7.0), and the mixture was sonicated in water for 10 min and stirred for 12 h under 4 1C. Abundant ADH was immobilized on the PDDA-protected GSs surface via electrostatic interaction. In addition, the resulting mixture was centrifuged at 12,000 rpm for 10 min and washed with double distilled water to remove the unbounded ADH. Finally, the obtained ADH-GSs bionanocomposite was redispersed in 1 mL distilled water and stored at 4 1C for further use. The morphology of ADH-GSs bionanocomposite was also studied by SEM and the result is shown in Fig. S1B (see Supplementary materials S1). 2.4. Preparation of secondary TB aptamer (TBA II): Hemin/Gquadruplex labeled AuNPs-ADH-GSs bionanocomposite First, 0.2 mL gold nanoparticles (AuNPs) colloid which was prepared according to the literature (Grabar et al., 1995) was added into 0.5 mL ADH-GSs bionanocomposite and stirred for 12 h under 4 1C. As a result, amount of AuNPs were immobilized onto the ADH-GSs bionanocomposite through the interaction between AuNPs and the NH2 in ADH. The AuNPs-ADH-GSs bionanocomposite was then obtained by centrifugation and washing with double distilled water. Second, the mixture of 1 mL TBA (0.25 μM) and 0.5 mg hemin was stirred for 2 h to form the hemin/ G-quadruplex structure according to our previous work (Yuan et al., 2013). The resulting hemin/G-quadruplex was subsequently added into the above AuNPs-ADH-GSs bionanocomposite and stirred under 4 1C for 12 h. Finally, 0.1 mL BSA (1%) was added into the solution for blocking the remaining active sites with 40 min stirring. TBA II was obtained by centrifuging, washing with double distilled water and it was stored at 4 1C when not in use. The preparation process of the TBA II was shown in Scheme 1A. For investigating the important role of graphene and AuNPs in the proposed aptasensor, hemin/G-quadruplex labled AuNPs-ADH bionanocomposite was also prepared in a similar way without adding graphene. Besides, hemin/G-quadruplex labeled ADH and
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hemin/G-quadruplex labeled ADH-GSs bionanocomposites were synthesized respectively with the method of carbodiimide activation reported before (Jordan et al., 2011; Yang et al., 2014): with EDC (2 mg/mL) serving as coupling agent between a carboxyl group on ADH and an amine group on hemin/G-quadruplex, 1 mL hemin/G-quadruplex solution prepared before was mixed with 0.5 mL ADH solution (2 mg/mL) and 0.5 mL prepared ADH-GSs solution, respectively. After stirring for 2 h under 4 1C, the resulting mixtures were centrifuged, washed with double distilled water and dispersed in 0.5 mL PBS buffer (pH 7.0) before use.
nonspecific binding effects. TB (20 μL) standard solutions with different concentrations were then incubated on the resulting electrode surface for 45 min at room temperature. At last, 20 μL TBA II was dropped onto the electrode surface for 1 h and the proposed aptasensor was obtained for electrochemical measurement.
2.5. Fabrication process of the sandwich-type electrochemical aptasensor
In order to characterize the modified process of the sensing surface, CV experiments of the proposed aptasensor were performed in 5 mM [Fe(CN)6]3 /4 solution at 100 mV/s scan rate, and the results are displayed in Fig. 1. Clearly, the GCE showed a well-defined redox peak of [Fe(CN)6]3 /4 (curve a). Upon the electrodeposition of HAuCl4 (1%) to the GCE, the peak current apparently increased (curve b), owing to the superior conductivity ability of the gold nanoparticles. The peak current dramatically decreased after the incubation of TBA I on the electrode surface (curve c), indicating the successful modification of TBA I and the increase of the steric hindrance. After incubating with BSA, the decreased peak current was also observed (curve d). Additionally, the peak current further decreased after the incubation with TB (curve e), accounting for the successful specific binding between TB and TBA I which retard the electron transfer tunnel.
The fabrication process of the sandwich-type aptasensor was illustrated in Scheme 1B. Prior to use, the GCE was carefully polished to a mirror-like surface with 0.3 μm and 0.05 μm alumina powder separately. After that, the polished GCE was sonicated in water and rinsed with double distilled water thoroughly. The cleaned GCE was immediately soaked in HAuCl4 solution (1%) and electrodeposited at 0.2 V for 30 s to introduce the gold nanoparticles layer (dep-Au). The 20 μL amino-terminated thrombin aptamer buffer (TBA I, 0.25 μM) was spread onto the electrode surface with 16 h incubation at room temperature. In addition, the modified electrode was incubated with 20 μL BSA solution (1%) for 40 min to block the remaining active sites and eliminate the
3. Results and discussion 3.1. Characterizations of the proposed electrochemical aptasensor
Scheme 1. The preparation process of the second thrombin aptamer (A) and the sandwich-type electrochemical aptasensor (B).
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Fig. 1. CVs of the stepwise modified aptasensor in 5 mM [Fe(CN)6]3 /4 solution with a scan rate of 100 mV/s: (a) GCE, (b) dep-Au/GCE, (c) TBA I/dep-Au/GCE, (d) BSA/TBA I/dep-Au/GCE, (e) TB/BSA/TBA I/dep-Au/GCE.
3.2. Signal amplified strategy of the aptasensor and the electron transfer of hemin The signal amplification strategy was accomplished by the corporate catalysis of ADH-GSs bionanocomposite and hemin/ G-quadruplex. It was compared by detecting the DPV signals before and after the addition of 80 μL NAD þ (0.25 mM) and 80 μL ethanol into the electrolytic cell (1 mL PBS, pH 7.0), and the results are displayed in Fig. 2A. As could be seen, no electrochemical signal was observed as the aptasensor was incubated with only 15 nM TB without TBA II (curve a). However, after incubation of 15 nM TB and 20 μL TBA II, the proposed aptasensor showed an increased current response (curve b), which originated from the successful modification of TBA II. After the addition of 80 μL absolute ethanol into the electrolytic cell (1.0 mL PBS, pH 7.0) containing 80 μL NAD þ (0.25 mM), obvious increase of the current response could be observed (curve c), owing to the corporate catalysis of ADH and hemin/G-quadruplex completed as follows: First, ADH could effectively catalyze the ethanol being oxidized into acetaldehyde with the reduction of NAD þ into NADH. Then, the hemin/G-quadruplex acted as the oxidase of NADH, assisting the oxidation of NADH to NAD þ with the produce of H2O2 in the presence of dissolved O2. Simultaneously, hemin/ G-quadruplex as HRP-mimicking DNAzyme catalyzed the reduction of H2O2. The above catalysis strategy promoted the redox reaction of hemin, achieving the amplified electrochemical signal. For demonstrating the electron transfer of hemin, two different TBA II (TBA labeled AuNPs-ADH-GSs and hemin/G-quadruplex labeled AuNPs-ADH-GSs bionanocomposites) were prepared and studied. The electron transfer of hemin was investigated by detecting the DPV signals after the incubation of the above mentioned bionanocomposites in PBS (pH 7.0) containing 80 μL NAD þ (0.25 mM) and 80 μL absolute ethanol. As shown in Fig. 2B, the electrochemical signal was hardly observed when the aptasensor was incubated with TBA labeled AuNPs-ADH-GSs bionanocomposite (curve a). Nevertheless, a distinct electrochemical signal was found after the aptasensor was incubated with hemin/G-quadruplex labeled AuNPs-ADH-GSs bionanocomposite (curve b), stemming from the electron transfer of hemin. These results indicated the electrochemical signal of the proposed aptasensor was attributed to electron transfer of hemin. 3.3. Electrochemical signal comparison of different types of TBA II In order to demonstrate the crucial role of graphene and AuNPs in the proposed strategy, control experiments were carried out by using four different TBA II bionanocomposites (hemin/G-quadruplex labeled
Fig. 2. (A) DPV curves of the aptasensor in three different conditions: (a) without TBA II in pH 7.0 PBS, (b) with TBA II in pH 7.0 PBS, and (c) with TBA II in pH 7.0 PBS containing 80 μL NAD þ (0.25 mM) and 80 μL absolute ethanol. (B) DPV curves of the aptasensor incubated with two bionanocomposites of (a) TBA labeled AuNPsADH-GSs and (b) hemin/G-quadruplex labeled AuNPs-ADH-GSs in pH 7.0 PBS containing 80 μL NAD þ (0.25 mM) and 80 μL absolute ethanol.
ADH, AuNPs-ADH, ADH-GSs and AuNPs-ADH-GSs, respectively). As could be seen from Fig. 3, the aptasensor with hemin/G-quadruplex labeled AuNPs-ADH-GSs bionanocomposite (Fig. 3D) showed much greater reduction peak current and electrochemical signal change in comparison with that obtained by the aptasensor with hemin/Gquadruplex labeled ADH, AuNPs-ADH, and ADH-GSs bionanocomposites (Fig. 3A–C), respectively. The results indicated the remarkable amplified performance of the proposed TBA II, which may ascribe to the following reasons: First, the employment of graphene with large surface area and excellent conductivity could not only improve the immobilized amount of ADH, AuNPs and hemin/G-quadruplex, but also enhance the catalysis efficiency of ADH and hemin/G-quadruplex. Second, AuNPs with good conductivity and biocompatibility could greatly improve the conductivity of ADH-GSs, provide more active sites for the immobilization of hemin/G-quadruplex and promote the electron transfer of hemin obviously, which further resulted in an amplified electrochemical signal. 3.4. Calibration curves of the proposed aptasensor Under the optimized experiment conditions (see Supplementary materials S2), the proposed aptasensor was incubated with a series of TB standard solution with various concentrations and the corresponding DPVs were recorded. As shown in Fig. 4A, the DPV current response was quite low in the presence of 0 nM TB, indicating the negligible unspecific binding. Besides, the DPV
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Fig. 3. DPV curves of the different sandwich format aptasensor in the absence (a) and in the presence (b) of 80 μL NAD þ (0.25 mM) and 80 μL absolute ethanol in 1 mL PBS (pH 7.0) by using different TBA II: (A) Hemin/G-quadruplex labeled ADH bionanocomposite. (B) Hemin/G-quadruplex labeled AuNPs-ADH bionanocomposite. (C) Hemin/Gquadruplex labeled ADH-GSs bionanocomposite. (D) Hemin/G-quadruplex labeled AuNPs-ADH-GSs bionanocomposite.
response increased with the increasing concentration of TB according to the typical sandwich mechanism. As a result, the electrochemical signal increased linearly with the logarithm of TB concentration (Fig. 4B) with a linear range from 1 pM to 50 nM. The linear equation was I¼ 2.059logc–11.54 (R¼0.9947), and the detection limit was 0.3 pM (defined as S/N¼ 3). The detection limit of the proposed TB aptasensor was comparable or even better than those of other reported methods and sandwich-type aptasensors for TB detection (see Supporting information Tables S1 and S2). The results should ascribe to the employment of ADH-GSs for great improvement of immobilization amount of ADH, the excellent catalytic ability of ADH and hemin/G-quadruplex simultaneously acted as NADH oxidase and HRP-mimicking DNAzyme. 3.5. Specificity, reproducibility, and stability of the aptasensor In the study, the specificity of the proposed aptasensor was investigated by challenging it against other possible interferences. Therefore, some other interfering substances such as L-cys, BSA, human IgG, and hemoglobin (Hb) were examined under the same experiment conditions. As shown in Fig. 5, no significant change of the current response could be observed in the detection of L-cys (100 nM), BSA (100 nM), human IgG (100 nM), Hb (100 nM), compared with that of the blank test. On the contrary, high current response was obtained in the presence of the target TB (15 nM) and its mixture with the above four interferences (100 nM). The results demonstrated the high specificity of the proposed aptasensor for TB detection. The reproducibility of the proposed aptasensor was evaluated by the following method: Five of the proposed aptasensors were incubated with 15 nM TB and detected under the same experiment conditions. The aptasensors exhibited similar electrochemical signals with a relative standard deviation (RSD) of 5.5%. In addition, the
same proposed aptasensor was used to detect TB (15 nM) for five times, a RSD of 7.4% was obtained. The results illustrated the acceptable reproducibility of the proposed aptasensor. The stability of the proposed aptasensor was demonstrated by long-term storage assay. The electrochemical signal retained 91.7% of its initial current after 15 days of storage at 4 1C, indicating the sufficient stability of the aptasensor for the TB detection. 3.6. Analytical application of the proposed aptasensor As a kind of serine protease, TB can convert soluble fibrinogen to insoluble strands of fibrin and catalyze many coagulationrelated reactions, resulting in its important role in clinic application (Rahman et al., 2009). Therefore, quantitative determination of TB is very important. In order to assess the reliability of the proposed aptasensor for TB detection, the practical applicability of the proposed aptasensor was investigated by adding different concentrations of TB into the 10-fold-diluted human real serum samples obtained from the Ninth People's Hospital (Chongqing, China). As indicated from Table S3 (see the Supporting information), the recovery and the RSDs were ranging from 92% to 108% and 2.3% to 5.7%, respectively. The results demonstrated the excellent promise for the detection of TB in real biological samples.
4. Conclusions In this study, a pseudo triple-enzyme cascade amplified aptasensor for TB detection based on hemin/G-quadruplex as signal label was successfully constructed. The combination of ADH-GSs bionanocomposite with hemin/G-quadruplex realized the pseudo triple-enzymatic and progressive amplification of electrochemical
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HRP-mimiking DNAzyme, giving rise to the electrochemical signal amplification. The aptasensor showed wide linear range, good specificity, satisfying reproducibility and sensitivity for the thrombin detection, providing a promising way for the determination of TB in clinical applications.
Acknowledgements This work was supported by the National Natural Science Foundation (NNSF) of China (21075100), State Key Laboratory of Electroanalytical Chemistry (SKLEAC2010009), High Technology Project Foundation of Southwest University (XSGX02), and the Postgraduate Science and Technology Innovation Program of Southwest University, China (Grant no. KB2011011).
Appendix A. Supplementary materials Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.11.036. References
Fig. 4. (A) DPV curves of the proposed aptasensor with different TB concentrations (0–50 nM) in 1 mL pH 7.0 PBS containing 80 μL NAD þ (0.25 mM) and 80 μL absolute alcohol. (B) Calibration curve of I vs. the logarithm of TB concentrations range from 1 pM to 50 nM under the optimized experiment conditions. The error bars indicated the standard deviation of three time measurements.
Fig. 5. Specificity of the proposed TB aptasensor against different interferences: (a) blank solution (0 nM TB), (b) 100 nM L-cys, (c) 100 nM BSA, (d) 100 nM IgG, (e) 100 nM Hb, (f) 15 nM TB, (g) 15 nM TBþ 100 nM L-cysþ100 nM BSAþ 100 nM IgGþ 100 nM Hb. The error bars indicated the standard deviation of three time measurements.
signal: In the presence of NAD þ and ethanol, the abundant ADH effectively catalyzed the oxidation of ethanol into acetaldehyde, coupling with the reduction of NAD þ into NADH. Then, hemin/ G-quadruplex simultaneously served as NADH oxidase and
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A pseudo triple-enzyme cascade amplified aptasensor for thrombin detection based on hemin/G-quadruplex as signal label Huayu Yi, Wenju Xu n, Yali Yuan, Lijuan Bai, Yongmei Wu, Yaqin Chai, Ruo Yuan n Key Laboratory on Luminescence and Real-Time Analysis, School of Chemistry and Chemical Engineering, Southwest University, The Key Laboratory of Eco-environments in Three Gorges Reservoir Region, Chongqing 400715, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 25 July 2013 Received in revised form 7 November 2013 Accepted 7 November 2013 Available online 18 November 2013
In this work, a pseudo triple-enzyme cascade amplified electrochemical aptasensor based on hemin/Gquadruplex as signal label for thrombin (TB) was constructed and the amplified electrochemical signal was achieved by the corporate catalysis of alcohol dehydrogenase-graphene sheets (ADH-GSs) bionanocomposite and hemin/G-quadruplex, which simultaneously acted as NADH oxidase and HRP-mimicking DNAzyme. Through “sandwich” reaction, hemin/G-quadruplex labeled gold nanoparticles-ADH-GSs bionanocomposite (AuNPs-ADH-GSs) was captured on electrode surface and thus obtained the electrochemical signal. After the addition of ethanol into the electrolytic cell, ADH availably catalyzed the oxidation of ethanol with the reduction of NAD þ to NADH. Then, hemin/G-quadruplex as NADH oxidase catalyzed the oxidization of NADH, accompanying with the production of H2O2. Simultaneously, hemin/Gquadruplex as HRP-mimicking DNAzyme catalyzed the reduction of the generated H2O2. Such a catalysis strategy greatly promoted the electron transfer of hemin and resulted in the specific enhancement of electrochemical signal. The proposed TB aptasensor achieved a linear range of 1 pM–50 nM with a detection limit of 0.3 pM (defined as S/N¼3). In addition, it showed satisfying stability and reproducibility, good specificity and sensitivity, indicating promising application for the detection of various proteins in clinical analysis. & 2013 Elsevier B.V. All rights reserved.
Keywords: Pseudo triple-enzyme Aptasensor Thrombin Alcohol dehydrogenase-graphene Hemin/G-quadruplex
1. Introduction Electrochemical aptamer-based sensors (aptasensors), based on the binding-induced conformational changes of redox-tagged and surface-confined aptamers (Zuo et al., 2007), have played an important role in the detection of proteins and small molecules (Wu et al., 2012; Cheng et al., 2012; Du et al., 2009), owing to the superior advantages, such as simple instrumentation, low cost, fast response, portability and high sensitivity (Liu et al., 2012; Chen et al., 2012). The aptasensors for this usage often involve in some redox mediators labeling, such as ferrocene (Liu et al., 2010), methylene blue (Fu et al., 2011), thionine (Tang et al., 2012) and so on. Alternatively, hemin referred to iron-containing porphyrin is the active center of hemoglobin (Kaneko et al., 2013; Cao et al., 2012) and can also offer electrochemical signal, which provides its possibility for constructing aptasensor. On the basis of this, an electrochemical thrombin (TB) aptasensor based on the electron transfer of hemin has been reported with a detection limit of 0.1 nM (Jiang et al., 2013). Besides, hemin also possesses excellent peroxidase activity when it forms the hemin/G-quadruplex structure via intercalating hemin into a guanine-rich single-stranded
n
Corresponding authors. Tel./fax: þ86 23 68252277. E-mail address: [email protected] (W. Xu).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.11.036
DNA with unique G-quadruplex structure (Meng et al., 2013). With advantages of good stability, inexpensive production and facile self-replication (Zhang et al., 2011; Chen et al., 2009), hemin/Gquadruplex structure has been used as peroxidase mimetic for detecting proteins (Li et al., 2008), metal ions (Li et al., 2010), small molecules (Li et al., 2007), and DNAs (Xiao et al., 2004). In addition, Golub and coworkers have proved that the hemin/ G-quadruplex structure can simultaneously act as horseradish peroxidase (HRP)-mimicking DNAzyme and NADH oxidase (Golub et al., 2011). Inspired by this property, an amplified TB electrochemical aptasensor was successfully fabricated by using the pseudobienzyme ability of hemin/G-quadruplex in our previous work (Yuan et al., 2012). Nevertheless, the combination of hemin/G-quadruplex as signal label with its remarkable catalytic performance has been received little attention in the field of electrochemical aptasensor. In recent years, multiple nanomaterials with unique electrical, physical and chemical properties have emerged for fabricating signal amplified biosensors (Guo et al., 2011; Li et al., 2012; He et al., 2011). Among them, graphene is widely used in biosensor applications with merits of flexibility, large surface area, excellent electrical conductivities and good mechanical properties (Chen et al., 2011; Lee et al., 2010). Thus, it is capable for graphene to immobilize a large amount of nanoparticles or enzymes (Zhang et al., 2011; Zeng et al., 2010), providing a promising approach for
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signal amplification in electrical biosensing (Bai et al., 2012). Additionally, the features of graphene endow the ability for facilitating electron transfer of redox mediators, which paves the way for fabrication of sensitive electrochemical aptasensors. With above advantages, graphene was employed as an enzyme immobilized platform in this work. Firstly, we prepared an alcohol dehydrogenase-graphene sheets (ADH-GSs) bionanocomposite, followed by assembly of plentiful gold nanoparticles (AuNPs) through the interaction between AuNPs and NH2 groups in ADH. In this way, a large amount of ADH and AuNPs could be immobilized on the graphene sheet with large surface area. Moreover, AuNPs could improve the conductivity of ADH-GSs bionanocomposite and further act as the platform for immobilizing thrombin aptamer (TBA). The obtained AuNPs-ADH-GSs bionanocomposite was then labled with hemin/G-quadruplex formed by intercalating hemin into the amino-terminated TBA. Finally, the hemin/G-quadruplex labeled AuNPs-ADH-GSs bionanocomposite (secondary thrombin aptamer: TBA II) with good redox and electrocatalytic ability was formed. Through “sandwich” reaction, TBA II was captured on the electrode surface, resulting in an electrochemical signal. Therefore, a pseudo triple-enzyme cascade amplified aptasensor was successfully designed for TB detection based on the corporate catalysis of ADH-GSs bionanocomposite and hemin/G-quadruplex, which simultaneously acted as NADH oxidase and HRP-mimicking DNAzyme. In the presence of ethanol and NAD þ , ADH effectively catalyzed the oxidation of ethanol to acetaldehyde, coupling with the production of NADH. Then, hemin/G-quadruplex acted as NADH oxidase, assisting the oxidation of NADH accompanying with the generation of H2O2 in the presence of dissolved O2. As HRPmimicking DNAzyme, hemin/G-quadruplex simultaneously catalyzed the reduction of H2O2. Finally, the electrochemical signal was amplified. The proposed aptasensor showed good sensitivity for quantitative determination of TB, which indicated a promising strategy for sensitive, simple detection of proteins in clinical applications.
2. Experimental 2.1. Materials and reagents Hemin, alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae, β-nicotinamide adenine dinucleotide hydrate (NAD þ ), thrombin (TB), bovine serum albumin (BSA), human IgG, hemoglobin (Hb), gold chloride (HAuCl4) were obtained from Sigma-Aldrich Chem. Co. (St. Louis, MO, USA). Graphene oxide (GO) was obtained from Nanjing XianFeng Nano Co. (Nanjing, China). Poly(diallyldimethylammonium chloride) (PDDA) was obtained from Beijing Chemical Reagent Co. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was provided by Shanghai Medpep Co. (Shanghai, China). L-cysteine (L-cys) was purchased from Kangan Amino Acid Company (Shanghai, China). Tris-hydroxymethylaminomethane hydrochloride (Tris) was purchased from Roche (Switzerland). Thrombin aptamer (TBA): 5′-NH2-(CH2)6-GGTTGGTGTGGTTGG-3′ was purchased from Sangon Biotech (Shanghai) Co., Ltd. 0.1 M PBS (pH 7.0) containing 10 mM Na2HPO4, 10 mM NaH2PO4 and 2 mM MgCl2 was used as working buffer solution, 20 mM Tris–HCl buffer (pH 7.4) containing 140 mM NaCl, 5 mM KCl, 1 mM CaCl2 and 1 mM MgCl2 was used as binding buffer solution. All other chemicals were of reagent grade and used as received. Double distilled water was used throughout the study. 2.2. Apparatus and measurements Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed with a CHI 660D electrochemical workstation (Shanghai Chenhua instrument, China). The scanning
electron micrographs were taken with scanning electron microscope (SEM, S-4800, Hitachi). All electrochemical experiments were conducted on a three-electrode system with a platinum wire as auxiliary electrode, a saturated calomel as reference electrode (SCE) and a bare or modified glassy carbon electrode (GCE, Ф ¼4 mm) as the working electrode. CV experiments were performed in 1.0 mL (5 mM) of [Fe(CN)6]3 /4 solution with the potential ranging from 0.2 V to 0.6 V. DPV measurements were carried out in 1.0 mL of 0.1 M PBS (pH 7.0) containing 80 μL NAD þ (0.25 mM) and 80 μL absolute ethanol. The scan rate was 100 mV/ s. The DPV measurements were taken with 4 mV of potential incremental, 50 mV of amplitude, 50 s of pulse width, and 0.0167 s of sample width with the potential ranging from 0.6 V to 0 V. 2.3. Preparation of ADH-graphene sheets (ADH-GSs) bionanocomposite Firstly, the PDDA-protected GSs with positive charge were synthesized according to the literature (Li et al., 2011) with a slight modification: A stable dispersion of exfoliated GO sheets (50 mL, 1 mg/mL) was mixed with PDDA (0.2 mL, 20%) and stirred for 30 min. Then, 0.5 mL hydrazine hydrate (80%) was added into the mixture with stirring for 24 h at 90 1C. Subsequently, the obtained black reaction product was centrifuged, washed with double distilled water and redispersed in water under mild sonication. The obtained PDDA-protected GSs were investigated by SEM observation, which is shown in Fig. S1A (see Supplementary materials S1). The ADH-GSs bionanocomposite was prepared as follows. Briefly, 0.5 mL PDDA-protected GSs prepared before was mixed with 0.5 mL ADH (pI ¼5.4) solution (2 mg/mL) prepared in PBS solution (pH 7.0), and the mixture was sonicated in water for 10 min and stirred for 12 h under 4 1C. Abundant ADH was immobilized on the PDDA-protected GSs surface via electrostatic interaction. In addition, the resulting mixture was centrifuged at 12,000 rpm for 10 min and washed with double distilled water to remove the unbounded ADH. Finally, the obtained ADH-GSs bionanocomposite was redispersed in 1 mL distilled water and stored at 4 1C for further use. The morphology of ADH-GSs bionanocomposite was also studied by SEM and the result is shown in Fig. S1B (see Supplementary materials S1). 2.4. Preparation of secondary TB aptamer (TBA II): Hemin/Gquadruplex labeled AuNPs-ADH-GSs bionanocomposite First, 0.2 mL gold nanoparticles (AuNPs) colloid which was prepared according to the literature (Grabar et al., 1995) was added into 0.5 mL ADH-GSs bionanocomposite and stirred for 12 h under 4 1C. As a result, amount of AuNPs were immobilized onto the ADH-GSs bionanocomposite through the interaction between AuNPs and the NH2 in ADH. The AuNPs-ADH-GSs bionanocomposite was then obtained by centrifugation and washing with double distilled water. Second, the mixture of 1 mL TBA (0.25 μM) and 0.5 mg hemin was stirred for 2 h to form the hemin/ G-quadruplex structure according to our previous work (Yuan et al., 2013). The resulting hemin/G-quadruplex was subsequently added into the above AuNPs-ADH-GSs bionanocomposite and stirred under 4 1C for 12 h. Finally, 0.1 mL BSA (1%) was added into the solution for blocking the remaining active sites with 40 min stirring. TBA II was obtained by centrifuging, washing with double distilled water and it was stored at 4 1C when not in use. The preparation process of the TBA II was shown in Scheme 1A. For investigating the important role of graphene and AuNPs in the proposed aptasensor, hemin/G-quadruplex labled AuNPs-ADH bionanocomposite was also prepared in a similar way without adding graphene. Besides, hemin/G-quadruplex labeled ADH and
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hemin/G-quadruplex labeled ADH-GSs bionanocomposites were synthesized respectively with the method of carbodiimide activation reported before (Jordan et al., 2011; Yang et al., 2014): with EDC (2 mg/mL) serving as coupling agent between a carboxyl group on ADH and an amine group on hemin/G-quadruplex, 1 mL hemin/G-quadruplex solution prepared before was mixed with 0.5 mL ADH solution (2 mg/mL) and 0.5 mL prepared ADH-GSs solution, respectively. After stirring for 2 h under 4 1C, the resulting mixtures were centrifuged, washed with double distilled water and dispersed in 0.5 mL PBS buffer (pH 7.0) before use.
nonspecific binding effects. TB (20 μL) standard solutions with different concentrations were then incubated on the resulting electrode surface for 45 min at room temperature. At last, 20 μL TBA II was dropped onto the electrode surface for 1 h and the proposed aptasensor was obtained for electrochemical measurement.
2.5. Fabrication process of the sandwich-type electrochemical aptasensor
In order to characterize the modified process of the sensing surface, CV experiments of the proposed aptasensor were performed in 5 mM [Fe(CN)6]3 /4 solution at 100 mV/s scan rate, and the results are displayed in Fig. 1. Clearly, the GCE showed a well-defined redox peak of [Fe(CN)6]3 /4 (curve a). Upon the electrodeposition of HAuCl4 (1%) to the GCE, the peak current apparently increased (curve b), owing to the superior conductivity ability of the gold nanoparticles. The peak current dramatically decreased after the incubation of TBA I on the electrode surface (curve c), indicating the successful modification of TBA I and the increase of the steric hindrance. After incubating with BSA, the decreased peak current was also observed (curve d). Additionally, the peak current further decreased after the incubation with TB (curve e), accounting for the successful specific binding between TB and TBA I which retard the electron transfer tunnel.
The fabrication process of the sandwich-type aptasensor was illustrated in Scheme 1B. Prior to use, the GCE was carefully polished to a mirror-like surface with 0.3 μm and 0.05 μm alumina powder separately. After that, the polished GCE was sonicated in water and rinsed with double distilled water thoroughly. The cleaned GCE was immediately soaked in HAuCl4 solution (1%) and electrodeposited at 0.2 V for 30 s to introduce the gold nanoparticles layer (dep-Au). The 20 μL amino-terminated thrombin aptamer buffer (TBA I, 0.25 μM) was spread onto the electrode surface with 16 h incubation at room temperature. In addition, the modified electrode was incubated with 20 μL BSA solution (1%) for 40 min to block the remaining active sites and eliminate the
3. Results and discussion 3.1. Characterizations of the proposed electrochemical aptasensor
Scheme 1. The preparation process of the second thrombin aptamer (A) and the sandwich-type electrochemical aptasensor (B).
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Fig. 1. CVs of the stepwise modified aptasensor in 5 mM [Fe(CN)6]3 /4 solution with a scan rate of 100 mV/s: (a) GCE, (b) dep-Au/GCE, (c) TBA I/dep-Au/GCE, (d) BSA/TBA I/dep-Au/GCE, (e) TB/BSA/TBA I/dep-Au/GCE.
3.2. Signal amplified strategy of the aptasensor and the electron transfer of hemin The signal amplification strategy was accomplished by the corporate catalysis of ADH-GSs bionanocomposite and hemin/ G-quadruplex. It was compared by detecting the DPV signals before and after the addition of 80 μL NAD þ (0.25 mM) and 80 μL ethanol into the electrolytic cell (1 mL PBS, pH 7.0), and the results are displayed in Fig. 2A. As could be seen, no electrochemical signal was observed as the aptasensor was incubated with only 15 nM TB without TBA II (curve a). However, after incubation of 15 nM TB and 20 μL TBA II, the proposed aptasensor showed an increased current response (curve b), which originated from the successful modification of TBA II. After the addition of 80 μL absolute ethanol into the electrolytic cell (1.0 mL PBS, pH 7.0) containing 80 μL NAD þ (0.25 mM), obvious increase of the current response could be observed (curve c), owing to the corporate catalysis of ADH and hemin/G-quadruplex completed as follows: First, ADH could effectively catalyze the ethanol being oxidized into acetaldehyde with the reduction of NAD þ into NADH. Then, the hemin/G-quadruplex acted as the oxidase of NADH, assisting the oxidation of NADH to NAD þ with the produce of H2O2 in the presence of dissolved O2. Simultaneously, hemin/ G-quadruplex as HRP-mimicking DNAzyme catalyzed the reduction of H2O2. The above catalysis strategy promoted the redox reaction of hemin, achieving the amplified electrochemical signal. For demonstrating the electron transfer of hemin, two different TBA II (TBA labeled AuNPs-ADH-GSs and hemin/G-quadruplex labeled AuNPs-ADH-GSs bionanocomposites) were prepared and studied. The electron transfer of hemin was investigated by detecting the DPV signals after the incubation of the above mentioned bionanocomposites in PBS (pH 7.0) containing 80 μL NAD þ (0.25 mM) and 80 μL absolute ethanol. As shown in Fig. 2B, the electrochemical signal was hardly observed when the aptasensor was incubated with TBA labeled AuNPs-ADH-GSs bionanocomposite (curve a). Nevertheless, a distinct electrochemical signal was found after the aptasensor was incubated with hemin/G-quadruplex labeled AuNPs-ADH-GSs bionanocomposite (curve b), stemming from the electron transfer of hemin. These results indicated the electrochemical signal of the proposed aptasensor was attributed to electron transfer of hemin. 3.3. Electrochemical signal comparison of different types of TBA II In order to demonstrate the crucial role of graphene and AuNPs in the proposed strategy, control experiments were carried out by using four different TBA II bionanocomposites (hemin/G-quadruplex labeled
Fig. 2. (A) DPV curves of the aptasensor in three different conditions: (a) without TBA II in pH 7.0 PBS, (b) with TBA II in pH 7.0 PBS, and (c) with TBA II in pH 7.0 PBS containing 80 μL NAD þ (0.25 mM) and 80 μL absolute ethanol. (B) DPV curves of the aptasensor incubated with two bionanocomposites of (a) TBA labeled AuNPsADH-GSs and (b) hemin/G-quadruplex labeled AuNPs-ADH-GSs in pH 7.0 PBS containing 80 μL NAD þ (0.25 mM) and 80 μL absolute ethanol.
ADH, AuNPs-ADH, ADH-GSs and AuNPs-ADH-GSs, respectively). As could be seen from Fig. 3, the aptasensor with hemin/G-quadruplex labeled AuNPs-ADH-GSs bionanocomposite (Fig. 3D) showed much greater reduction peak current and electrochemical signal change in comparison with that obtained by the aptasensor with hemin/Gquadruplex labeled ADH, AuNPs-ADH, and ADH-GSs bionanocomposites (Fig. 3A–C), respectively. The results indicated the remarkable amplified performance of the proposed TBA II, which may ascribe to the following reasons: First, the employment of graphene with large surface area and excellent conductivity could not only improve the immobilized amount of ADH, AuNPs and hemin/G-quadruplex, but also enhance the catalysis efficiency of ADH and hemin/G-quadruplex. Second, AuNPs with good conductivity and biocompatibility could greatly improve the conductivity of ADH-GSs, provide more active sites for the immobilization of hemin/G-quadruplex and promote the electron transfer of hemin obviously, which further resulted in an amplified electrochemical signal. 3.4. Calibration curves of the proposed aptasensor Under the optimized experiment conditions (see Supplementary materials S2), the proposed aptasensor was incubated with a series of TB standard solution with various concentrations and the corresponding DPVs were recorded. As shown in Fig. 4A, the DPV current response was quite low in the presence of 0 nM TB, indicating the negligible unspecific binding. Besides, the DPV
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Fig. 3. DPV curves of the different sandwich format aptasensor in the absence (a) and in the presence (b) of 80 μL NAD þ (0.25 mM) and 80 μL absolute ethanol in 1 mL PBS (pH 7.0) by using different TBA II: (A) Hemin/G-quadruplex labeled ADH bionanocomposite. (B) Hemin/G-quadruplex labeled AuNPs-ADH bionanocomposite. (C) Hemin/Gquadruplex labeled ADH-GSs bionanocomposite. (D) Hemin/G-quadruplex labeled AuNPs-ADH-GSs bionanocomposite.
response increased with the increasing concentration of TB according to the typical sandwich mechanism. As a result, the electrochemical signal increased linearly with the logarithm of TB concentration (Fig. 4B) with a linear range from 1 pM to 50 nM. The linear equation was I¼ 2.059logc–11.54 (R¼0.9947), and the detection limit was 0.3 pM (defined as S/N¼ 3). The detection limit of the proposed TB aptasensor was comparable or even better than those of other reported methods and sandwich-type aptasensors for TB detection (see Supporting information Tables S1 and S2). The results should ascribe to the employment of ADH-GSs for great improvement of immobilization amount of ADH, the excellent catalytic ability of ADH and hemin/G-quadruplex simultaneously acted as NADH oxidase and HRP-mimicking DNAzyme. 3.5. Specificity, reproducibility, and stability of the aptasensor In the study, the specificity of the proposed aptasensor was investigated by challenging it against other possible interferences. Therefore, some other interfering substances such as L-cys, BSA, human IgG, and hemoglobin (Hb) were examined under the same experiment conditions. As shown in Fig. 5, no significant change of the current response could be observed in the detection of L-cys (100 nM), BSA (100 nM), human IgG (100 nM), Hb (100 nM), compared with that of the blank test. On the contrary, high current response was obtained in the presence of the target TB (15 nM) and its mixture with the above four interferences (100 nM). The results demonstrated the high specificity of the proposed aptasensor for TB detection. The reproducibility of the proposed aptasensor was evaluated by the following method: Five of the proposed aptasensors were incubated with 15 nM TB and detected under the same experiment conditions. The aptasensors exhibited similar electrochemical signals with a relative standard deviation (RSD) of 5.5%. In addition, the
same proposed aptasensor was used to detect TB (15 nM) for five times, a RSD of 7.4% was obtained. The results illustrated the acceptable reproducibility of the proposed aptasensor. The stability of the proposed aptasensor was demonstrated by long-term storage assay. The electrochemical signal retained 91.7% of its initial current after 15 days of storage at 4 1C, indicating the sufficient stability of the aptasensor for the TB detection. 3.6. Analytical application of the proposed aptasensor As a kind of serine protease, TB can convert soluble fibrinogen to insoluble strands of fibrin and catalyze many coagulationrelated reactions, resulting in its important role in clinic application (Rahman et al., 2009). Therefore, quantitative determination of TB is very important. In order to assess the reliability of the proposed aptasensor for TB detection, the practical applicability of the proposed aptasensor was investigated by adding different concentrations of TB into the 10-fold-diluted human real serum samples obtained from the Ninth People's Hospital (Chongqing, China). As indicated from Table S3 (see the Supporting information), the recovery and the RSDs were ranging from 92% to 108% and 2.3% to 5.7%, respectively. The results demonstrated the excellent promise for the detection of TB in real biological samples.
4. Conclusions In this study, a pseudo triple-enzyme cascade amplified aptasensor for TB detection based on hemin/G-quadruplex as signal label was successfully constructed. The combination of ADH-GSs bionanocomposite with hemin/G-quadruplex realized the pseudo triple-enzymatic and progressive amplification of electrochemical
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HRP-mimiking DNAzyme, giving rise to the electrochemical signal amplification. The aptasensor showed wide linear range, good specificity, satisfying reproducibility and sensitivity for the thrombin detection, providing a promising way for the determination of TB in clinical applications.
Acknowledgements This work was supported by the National Natural Science Foundation (NNSF) of China (21075100), State Key Laboratory of Electroanalytical Chemistry (SKLEAC2010009), High Technology Project Foundation of Southwest University (XSGX02), and the Postgraduate Science and Technology Innovation Program of Southwest University, China (Grant no. KB2011011).
Appendix A. Supplementary materials Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.11.036. References
Fig. 4. (A) DPV curves of the proposed aptasensor with different TB concentrations (0–50 nM) in 1 mL pH 7.0 PBS containing 80 μL NAD þ (0.25 mM) and 80 μL absolute alcohol. (B) Calibration curve of I vs. the logarithm of TB concentrations range from 1 pM to 50 nM under the optimized experiment conditions. The error bars indicated the standard deviation of three time measurements.
Fig. 5. Specificity of the proposed TB aptasensor against different interferences: (a) blank solution (0 nM TB), (b) 100 nM L-cys, (c) 100 nM BSA, (d) 100 nM IgG, (e) 100 nM Hb, (f) 15 nM TB, (g) 15 nM TBþ 100 nM L-cysþ100 nM BSAþ 100 nM IgGþ 100 nM Hb. The error bars indicated the standard deviation of three time measurements.
signal: In the presence of NAD þ and ethanol, the abundant ADH effectively catalyzed the oxidation of ethanol into acetaldehyde, coupling with the reduction of NAD þ into NADH. Then, hemin/ G-quadruplex simultaneously served as NADH oxidase and
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