Analytica Chimica Acta 860 (2015) 70–76

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An exonuclease-assisted amplification electrochemical aptasensor of thrombin coupling “signal on/off” strategy Ting Bao, Wei Wen, Xiuhua Zhang, Shengfu Wang * Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Ministry of Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules & College of Chemistry and Chemical Engineering, Hubei University, Wuhan 430062, PR China

H I G H L I G H T S

G R A P H I C A L A B S T R A C T


MB and Fc were labeled on two oligonucleotides separately to produce dual signals.
Coupling “signal-on” and “signal-off” strategies.
Exonuclease-catalyzed target recycling amplified two signals significantly.

A R T I C L E I N F O

A B S T R A C T

Article history: Received 13 November 2014 Received in revised form 7 December 2014 Accepted 12 December 2014 Available online 17 December 2014

In this work, a dual-signaling electrochemical aptasensor based on exonuclease-catalyzed target recycling was developed for thrombin detection. The proposed aptasensor coupled “signal-on” and “signal-off” strategies. As to the construction of the aptasensor, ferrocene (Fc) labeled thrombin binding aptamer (TBA) could perfectly hybridize with the methylene blue (MB) modified thiolated capture DNA to form double-stranded structure, hence emerged two different electrochemical signals. In the presence of thrombin, TBA could form a G-quadruplex structure with thrombin, leading to the dissociation of TBA from the duplex DNA and capture DNA formed hairpin structure. Exonuclease could selectively digest single-stranded TBA in G-quadruplex structure and released thrombin to realize target recycling. As a consequence, the electrochemical signal of MB enhanced significantly, which realized “signal on” strategy, meanwhile, the deoxidization peak current of Fc decreased distinctly, which realized “signal off” strategy. The employment of exonuclease and superposition of two signals significantly improved the sensitivity of the aptasensor. In this way, an aptasensor with high sensitivity, good stability and selectivity for quantitative detection of thrombin was constructed, which exhibited a good linear range from 5 pM to 50 nM with a detection limit of 0.9 pM (defined as S/N = 3). In addition, this design strategy could be applied to the detection of other proteins and small molecules. ã 2014 Elsevier B.V. All rights reserved.

Keywords: Electrochemical aptasensor Dual-signaling Exonuclease Target recycling Thrombin

1. Introduction Thrombin is a serine protease which converts soluble fibrinogen into insoluble fibrin during the coagulation progress. It plays an important role in many crucial physiological and pathological

* Corresponding author. Tel.: +86 27 50865309; fax: +86 27 88663043. E-mail address: [email protected] (S. Wang). http://dx.doi.org/10.1016/j.aca.2014.12.027 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.

processes such as blood coagulation, thrombosis, inflammation and angiogenesis [1–4]. Thrombin concentration in blood varies from nanomolar to low micromolar levels [5], the concentration of thrombin is in connection with various coagulation abnormalities [6]. Therefore, quantitative detection of thrombin is significant in disease diagnosis and clinical practice [7]. So far, mass spectrometry [8], surface plasmon resonance [9] and fluorescence spectroscopy [10] have been used for the detection of thrombin. However, these methods are rather expensive, sophisticated and unsuitable

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for fast detection, while electrochemical methods have received lots of attention for the advantage of simpleness, fast response and in vivo detection. Aptamers are single-stranded DNA or RNA oligonucleotides, which are synthesized by systematic evolution of ligands by exponential enrichment (SELEX) technology [11,12]. They can specifically recognize and bind to lots of targets, including small molecules, proteins and cells [13–16]. Compared with antibody, aptamers have sorts of advantages such as good stability, low cost, easy modification, long-term storage and high resistance against denaturation [17]. Aptamer based biosensorhas manyapplications in food safety, pharmaceutical analysis, environmental monitoring and biochemical analysis [18–21]. For the great importance of thrombin, thrombin binding aptamer (TBA) is also been widely investigated [22]. TBA can bind to different epitopes of human a-thrombin and form a stable G-quadruplex structure with the excellent affinity and high selectivity towards thrombin, which has been widely used to construct biosensor for the detection of thrombin [23,24]. So far, two TBA has been founded: a 15 bases aptamer can bind to exosite I of thrombin, known as fibrinogen binding sites, and a 29 bases aptamer can bind to exosite II of thrombin, called heparin-binding aptamer. 29 bases aptamer has higher affinity towards thrombin than 15 bases aptamer [25,26]. In this work, 29 based TBA was chosen to construct proposed aptasensor. Generally, ferrocene (Fc) [27], methylene blue (MB) [28] and thionine [29] are applied to label aptamers to produce an electrochemical signal. The research of dual-signaling aptasensor is still a challenge [30]. Many signal amplification strategies have been used to improve the sensitivity of aptasensors, such as gold nanoparticles assisted amplification [31], DNAzyme assisted amplification [32], aptamer/ graphene (graphene oxide) nanocomplex assisted amplification [33–35], rolling circle amplification [36], hybridization chain reaction amplification [37], enzyme labeling amplification [38], exonuclease-catalyzed target recycling [39], and so on. Among them, exonuclease can selectively digest aptamer which binds to target and release the analyte for target recycling, providing an excellent method for sensitive detection in aptasensors [40]. RecJf exonuclease is processive single-stranded DNA specific enzyme, which functions unidirectionally at 50 -termini. It can degrade single-strand DNA in the direction of 50 ! 30 [41,42]. Exonuclease-catalyzed target recycling strategy amplifies the signal and improves the sensitivity of aptasensor significantly [43], which has been applied in the construction of aptasensor. However, most of aptasensor has only one signal, they are only “signal-on” or “signal-off” type, exonuclease can only enhance one signal. While the proposed aptasensor has two different signals, which can reach both “signal-on” and “signal-off” strategy, and exonuclease-catalyzed target recycling amplifies two different signals simultaneously. Herein, we proposed a “signal on/off” electrochemical aptasensor for thrombin detection based on exonuclease-catalyzed target recycling. To construct the aptasensor, two different signaling molecules (MB and Fc) were labeled on capture DNA and TBA separately to produce two different signals. TBA formed aptamer–thrombin complex with thrombin and released from electrode, which led to the decrease of Fc signal, while the capture DNA formed hairpin structure, the signal of MB was increased. The aptasensor coupled signal-on and signal-off strategies, exonuclease amplified two different signals simultaneously, improving the sensitivity of the aptasensor significantly.

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hydrochloride (TCEP) were purchased from Sigma–Aldrich Chemical Co. (USA). RecJf exonuclease was obtained from New England Biolabs Ltd. (Beijing, China). BSA and Myoglobin were ordered from Aladdin Chemistry Co., Ltd. (China). Oligonucleotides were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). All other reagents were of analytical reagent grade. All solutions were prepared using ultrapure water (18.25 MV cm1) produced by Aquapro Ultra-pure water system (China). 20 mM tris-HCl buffer (pH 7.4) containing 140 mM NaCl, 5 mM KCl and 1 mM MgCl2 was used to dissolve thrombin and DNA oligonucleotides. The sequence of oligonucleotides were as follows: capture DNA: 50 -HS-(CH2)6-AGTCACCCCAACCTGCCCTACCACGGACT-MB-30 . The complementary four bases (underline showed) at both its 50 and 30 ends to make the capture DNA form a stem-loop structure. TBA: 50 -AAAAGTCCGTGGTAGGGCAGGTTGGGGTGACT-Fc-30 . Three bases were added (underline showed) at the 50 end of the thrombin binding aptamer, in order to make RecJf exonuclease recognize the single strand in aptamer–thrombin complex more easily. 2.2. Apparatus All electrochemical characterizations including cyclic voltammetry (CV), differential pulse voltammetric (DPV) and electrochemical impedance spectroscopic (EIS) measurements were carried out on a CHI660C electrochemical workstation (Shanghai Chenhua Instrument, China). A three-electrode system composed of platinum wire as the auxiliary electrode, saturated calomel electrode (SCE) as reference electrode and a 2-mm-diameter gold electrode as working electrode was used in the experiment. All the electrodes were purchased from CH Instruments, Inc., CV and DPV were performed in 10 mM PBS (pH 7.4) with a voltage range from 0.6 V to 0.5 V. Ultrasonic cleaners (Branson2000, USA) and 320-S acidity meters (Mettler-Toledo, Switzerland) were used in this experiment. 2.3. Preparation of the electrochemical aptasensor The bare gold electrode (GE) was polished with 0.05 mm of Gamma-alumina powder and ultrasonic cleaning in water and ethanol separately. Then, the GE was electrochemical cleaned through scanned in 0.5 M H2SO4 by cyclic voltammetry for 10 scans from 0.3 V to 1.5 V at a scan rate of 100 mV s1. After drying with nitrogen, the electrode was immediately dipped into 50 mL capture DNA (0.5 mM) containing 0.2 mM TCEP for 12 h at room temperature (prior to use, the capture DNA was heated to 90  C for 10 min and then immediately cooled down in ice-bath. In this way, the capture DNA kept single strand structure). Next, the modified electrode was soaked into 1 mM MCH for 1 h to block the free sites on GE. Then, the electrode was immersed into 50 mL TBA (1 mM) for 2 h at 37  C. After that, the electrode was incubated in 50 mL of the mixture of different concentration of thrombin containing 0.03 U mL1 RecJf exonuclease in 1 NEBuffer 2 (50 mM NaCl, 10 mM trisHCl, 10 mM MgCl2, 1 mM DTT, pH 7.9) for 90 min at 37  C. Finally, the electrode was used for measurement in 10 mM PBS (pH 7.4). After each step, the electrode was rinsed with PBS (pH 7.4) to remove the adsorbate. 3. Results and discussion

2. Experimental 3.1. Design strategy of the aptasensor 2.1. Materials and reagents Thrombin (95%, SDS-PAGE), 6-mercapto-1-hexanol (MCH), immunoglobulin G (IgG) and tris(2-carboxyethy)phosphine

As shown in Fig. 1, capture DNA firstly self-assembled onto the electrode surface, and the Fc labeled TBA perfectly hybridized with capture DNA. The formation of double-stranded structure made

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the MB relatively far from the GE surface and Fc get close to the GE. As a result, the peak current of MB was small and that of Fc was obvious (curve a). With the employment of thrombin and exonuclease, TBA formed stable aptamer–thrombin G-quadruplex structure with thrombin and released from GE. The double-strand DNA was destroyed and capture DNA changed to hairpin structure because of four complementary bases at both ends. RecJf exonuclease selectively degraded TBA in aptamer–thrombin complex in the direction of 50 ! 30 , thrombin was released into the solution for target recycling. As a result, the peak current of MB increased significantly, which realized signal-on strategy, while the peak current of Fc significantly decreased, which realized signal-off strategy (curve b). 3.2. Electrochemical characterization of the aptasensor The aptasensor was characterized by EIS and CV measurements [44]. In EIS measurement was performed in 0.5 mM [Fe(CN)6]3/4 containing 0.1 M KCl, [Fe(CN)6]3/[Fe(CN)6]4 was utilized as the redox probe and the semicircle diameter equated the electrontransfer resistance, Ret. As shown in Fig. 2A, bare GE exhibited an almost straight line (curve a), indicated the bare GE had a low resistance and allowed fast electron-transfer. After thiolated capture DNA self-assembled onto the electrode surface, the Ret increased significantly (curve b), because the oligonucleotides were non-electroactive, the resistance of electron-transfer was strong. The Ret kept increasing (curve c) when the aptasensor was blocked unoccupied sites by MCH, since the MCH was nonconductive. After hybridizing with TBA, the Ret was further increased (curve d), and it decresed observably after incubated with the mixture of 1 nM thrombin and 0.03 U mL1 RecJf exonuclease (curve e). Due to the formation of TBA–thrombin complex made the double-strand DNA unwinding, and exonuclease-catalyzed target recycling made the non-conductive

oligonucleotides on the GE surface decreased. These results were in line with CV measurements (Fig. 2B). The CV of bare GE had the highest peak current (curve a). Capture DNA on the GE made the CV peak current decrease due to the increase of the electron-transfer resistance (curve b). MCH blocked electrode made the peak current decrease further (curve c). The peak current decreased when TBA hybridized with capture DNA (curve d), and it increased with the addition of thrombin and exonuclease (curve e). In addition, DPV response of different modified electrodes were investigated to verify the construction process of the aptasensor. As shown in Fig. 2C, the capture DNA modified GE which blocked by MCH showed a dramatic deoxidization peak of MB at about 0.25 V (curve a). After hybridizing with sufficient Fc tagged TBA, the aptasensor produced a significant decrease of the MB DPV response, meanwhile, the Fc DPV response increased at about 0.29 V (curve b). It indicated the perfect hybridization of capture DNA with TBA and the dual-signaling aptasensor successfully constructed. When electrode was incubated with 1 nM thrombin containing 0.03 U mL1 RecJf exonuclease solution, the DPV current of Fc was decreased while MB DPV response increased (curve c), owing to TBA binding with thrombin which dissociated from the electrode and exonuclease-assisted target recycling, while the single-stranded capture probe transformed into hairpin structure. These results clearly indicated that the developed aptasensor coupled signal-on and signal-off strategies to detect thrombin. 3.3. Amplification performance of the proposed aptasensor In order to investigate whether the employed of RecJf exonuclease could significantly amplify the signal, DPV responses in the absence and presence of exonuclease were detected. As shown in Fig. 3, without thrombin and exonuclease (blank), MB deoxidization peak current was small and that of Fc was big (curve a). With the introduction of 1 nM thrombin, the MB signal

[(Fig._1)TD$IG]

Fig. 1. Schematic diagram of the exonuclease-assisted amplification electrochemical aptasensor of thrombin coupling “signal on/off” strategy.

[(Fig._2)TD$IG]

T. Bao et al. / Analytica Chimica Acta 860 (2015) 70–76

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Fig. 2. EIS (A) and CVs (B) of the stepwise modified aptasensor in 5 mM [Fe(CN)6]3/4 containing 0.1 M KCl: (a) GE, (b) capture DNA/GE, (c) MCH/capture DNA/GE, (d) TBA/ MCH/capture DNA/GE, and (e) TBA/MCH/capture DNA/GE incubated with 1 nM thrombin and 0.03 U mL1 exonuclease. The scan rate was 100 mV s1. (C) The DPV response curves of different modified electrodes: (a) MCH/capture DNA/GE, (b) TBA/MCH/capture DNA/GE, and (c) TBA/MCH/capture DNA/GE incubated with 1 nM thrombin and 0.03 U mL1 exonuclease.

increased and the Fc signal decreased (curve b). With the addition of the RecJf exonuclease, the increase of MB DPV response and the decrease of Fc DPV response became more apparently (curve c), because of the RecJf exonuclease could selectively degrade TBA in aptamer–thrombin G-quadruplex and release thrombin to participate target recycling to amplified the signal. 3.4. Optimization of experimental conditions The concentration proportion of capture DNA and TBA could impact the hybridization effect. To make the hybridization sufficiently, different concentration proportion of capture DNA and TBA (1:1, 2:3, 1:2, 1:3) were discussed. Seen from Fig. 4A, when the concentration proportion of them was 1:2, the DPV current of MB and that of Fc reached saturation, indicating the hybridization process had been fully performed. Therefore, the optimal concentration proportion of capture DNA and TBA was chosen as 1:2. The incubation time of aptasensor with thrombin and RecJf exonuclease had great influence on the exonuclease digestion effect, which was very important to the amplification performance of the aptasensor. The DPV responses for 1 nM thrombin and 0.03 U mL1 exonuclease with different incubation time was detected. As shown in Fig. 4B, it is clearly observed that the DPV responses of MB increased (curve a) and that of Fc decreased (curve b) with the increase of incubation time and both of them leveled off at 90 min. Therefore, 90 min was chosen as the optimum incubation time.

3.5. Analytical performance of the proposed aptasensor Under the optimized experimental conditions, the proposed aptasensor was incubated with a series concentration of thrombin containing 0.03 U mL1 RecJf exonuclease and DPV response were recorded. As shown in Fig. 5, the DPV currents of MB increased and that of Fc decreased with the concentration of thrombin raised from 0 to 50 nM. The calibration plot was illustrated in the insert, it showed two calibration curves: the peak current change of MB

[(Fig._3)TD$IG]

Fig. 3. DPV response curves for 1 nM thrombin detection: (a) in blank, (b) in the absence of exonuclease, and (c) in the presence of 0.03 U mL1 exonuclease.

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[(Fig._4)TD$IG]

T. Bao et al. / Analytica Chimica Acta 860 (2015) 70–76

Fig. 4. (A) Optimization of the proportion of capture probe and TBA, four proportions were investigated: 1:1, 2:3, 1:2, 1:3. (B) Optimization of incubation time of the prepared aptasensor with the mixture of thrombin and exonuclease.

(DIMB) and that of Fc (DIFc) exhibited a good linear correlation to the logarithm of the concentrations of the thrombin ranging from 5 pM to 50 nM. The linear equation were:

3.6. Specificity and reproducibility of the aptasensor for thrombin detection

curve a :DIMB ðmAÞ ¼ 0:0732  0:0182lgcðnMÞðR ¼ 0:999Þ

(1)

curve b :DIFc ðmAÞ ¼ 0:0758 þ 0:0176lgcðnMÞðR ¼ 0:998Þ

(2)

The specificity of the proposed aptasensor was investigated by detecting several interfering proteins. Therefore, BSA, IgG, and Myoglobin were detected under the same experimental condition, the concentrations of them were 50 nM. Seen from Fig. 6, no significant current change was observed when the interfering proteins were added compared with blank test. However, 1 nM thrombin led to an obvious increase of MB DPV peak current and dramatic decrease of Fc DPV response. The result demonstrated the proposed aptasensor had high specificity to thrombin. The reproducibility of the aptasensor was investigated: five equally prepared electrodes were used to detect thrombin (1 nM). Five electrodes exhibited similar electrochemical responses and a relative standard deviation (RSD) of MB and Fc peak current were 4.4% and 6.4%. All the results manifested the proposed aptasensor had acceptable reproducibility.

The detection limits were 2.2 pM and 2.7 pM (S/N = 3). To amplify the electrochemical response, the sum of DIMB and DIFc was used as the response signal for the detection of thrombin, expressed it as DIsum (DIsum = DIMB  DIFc). The linear regression equation was adjusted to:

DIsum ðmAÞ ¼ 0:1504  0:0361lg cðnMÞ

(3)

The limit of detection (LOD) was 0.9 pM (S/N = 3). It was lower than 1.7 pM obtained by single-signaling aptasensor based on exonuclease-catalyzed target recycling [45].

[(Fig._5)TD$IG]

3.7. Analytical application of the aptasensor In order to evaluate the reliability of the proposed aptasensor for thrombin detection, recovery experiments were performed by standard addition methods in human serum. Different concentrations of thrombin were added into 1000-fold-diluted human serum samples. From Table 1, it showed that the recovery was from 93.1% to 104.5%, and the RSD was between 2.6% and 3.4%. These

[(Fig._6)TD$IG]

Fig. 5. DPV responses of the aptasensor incubated with the mixture of different concentration of thrombin and exonuclease (from a ! h: 0, 0.005, 0.05, 0.5, 1, 5, 25, 50 nM thrombin). The inset showed the calibration curve corresponding to the DPV responses of DIMB and DIFc with the logarithm of thrombin concentrations ranged from 5 pM to 50 nM under the optimized conditions.

Fig. 6. Selectivity of the aptasensor to thrombin, BSA, IgG, and myoglobin. Concentration of thrombin was 5 nM and others were 50 nM.

T. Bao et al. / Analytica Chimica Acta 860 (2015) 70–76 Table 1 Detection of thrombin added in human serum (n = 3) with the proposed aptasensor. Samples

Added (nM)

Found (nM)

Recovery (%)

RSD (%)

1 2 3

5 0.1 0.01

4.844 0.1045 0.00931

96.9 104.5 93.1

2.6 2.6 3.4

[15]

[16]

[17]

results indicated proposed aptasensor had a potential to detect thrombin in real biological samples.

[18]

4. Conclusion

[19]

In summary, an exonuclease-assisted amplification electrochemical aptasensor coupling “signal on/off” strategy for the detection of thrombin was successfully constructed. The MB labeled capture DNA acted as a “signal-on” probe, meanwhile the Fc labeled TBA worked as a “signal-off” probe. The employment of exonuclease-catalyzed target recycling and the superposition of two signals significantly improved the sensitivity of the proposed aptasensor. The aptasensor showed high sensitivity, good stability, acceptable reproducibility, wide detection range and a low detection limit for thrombin detection, which had a great promise for sensitive detection of thrombin in clinical diagnosis.

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