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Cleavage-based hybridization chain reaction for electrochemical detection of thrombin† Yuanyuan Chang, Yaqin Chai,* Shunbi Xie, Yali Yuan, Juan Zhang and Ruo Yuan* In the present work, we constructed a new label-free “inter-sandwich” electrochemical aptasensor for thrombin (TB) detection by employing a cleavage-based hybridization chain reaction (HCR). The designed single-stranded DNA (defined as binding DNA), which contained the thrombin aptamer binding sequence, a DNAzyme cleavage site and a signal reporter sequence, was first immobilized on the electrode. In the absence of a target TB, the designed DNAzymes could combine with the thrombin aptamer binding sequence via complementary base pairing, and then Cu2+ could cleave the binding DNA. In the presence of a target TB, TB could combine with the thrombin aptamer binding sequence to predominantly form an aptamer–protein complex, which blocked the DNAzyme cleavage site and prevented the binding DNA from being cleaved by Cu2+-dependent DNAzyme. As a result, the signal reporter sequence could leave the electrode surface to trigger HCR with the help of two auxiliary DNA single-strands, A1 and A2. Then, the electron mediator hexaammineruthenium (III) chloride ([Ru(NH3)6]3+) was embedded into the double-stranded DNA (dsDNA) to produce a strong electrochemical signal for the quantitative measurement of TB. For further amplification of the electrochemical signal, graphene reduced by dopamine (PDA-rGO) was introduced as a platform in this work. With this strategy, the
Received 22nd April 2014 Accepted 6th June 2014
aptasensor displayed a wide linearity in the range of 0.0001 nM to 50 nM with a low detection limit of 0.05 pM. Moreover, the resulting aptasensor exhibited good specificity and acceptable reproducibility
DOI: 10.1039/c4an00712c
and stability. Because of these factors, the fabrication protocol proposed in this work may be extended to clinical application.
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Introduction The functional nucleic acid DNAzymes (also known as catalytic DNA, deoxyribozymes, or DNA enzymes) are a kind of articial enzymes.1,2 In addition their function as catalysts, another attractive aspect of DNAzymes is that they have a metal-iondependent cleavage specic, partially complementary DNA substrate in the presence of metal ions.3 The special cleavage enzyme possesses the qualities of being relatively less expensive, easily labeled, highly stable and offering straightforward sequence specic recognition.4–6 Because of these properties, metal-ion-dependent DNAzymes, as cleavage enzymes, have exhibited surprising potential in biochemical reactions in recent years. For example, Gao's group successfully constructed a lead biosensor based on GR-5 lead-dependent DNAzyme.7 Zhang's group proposed a new electrochemical immunoassay protocol based on the cleavage of metal-ion-induced DNAzymes for the determination of protein with high sensitivity and Education Ministry Key Laboratory of Luminescent and Real-Time Analytical Chemistry, College of Chemistry and Chemical Engineering, Chongqing 400715, People's Republic of China. E-mail: [email protected]; [email protected]; Fax: +86-23-68253172; Tel: +86-23-68252277 † Electronic supplementary 10.1039/c4an00712c
information
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(ESI)
available.
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DOI:
specicity.8 Furthermore, Chen's group recently reported a novel method that chose Cu2+-dependent. DNAzymes as the cleavage enzyme to quantitatively measure protein-aptamer complexation on the surface by blocking DNAzyme cleavage activity.9 Therefore, metal-ion-dependent DNAzymes as a novel kind of cleavage enzymes provide a new path to develop various biosensors. The improvement of electrochemical biosensors with high sensitivity aims at reducing detection limits and amplifying the signal by using various methods.10 Thus, numerous DNA amplication technologies, including polymerase chain reaction (PCR),11 rolling circle amplication (RCA),12 strand displacement amplication (SDA)13 and hybridization chain reaction (HCR)14–16 have been developed. Among these methods, HCR is one of the most frequently used strategies, especially in the eld of electrochemical technology.17–20 HCR is a self-assembled and enzyme-free process that can perform a cascade of hybridization events with two single-stranded DNA (ssDNA) molecules to spontaneously form a long doublestranded DNA (dsDNA) molecule based on the energy of base pair formation under mild conditions14,21–23 in the presence of an initiator DNA. The formed dsDNA with a negative charge and stably rigid structure was an ideal carrier for loading numerous electrochemically active compounds via electrostatic adsorption
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or labeling.24 For instance, the positively charged electron mediator hexaammineruthenium (III) chloride ([Ru(NH3)6]3+) can automatically embed into dsDNA via electrostatic adsorption,25 and the process avoids the complex labeling steps by enhancing electrochemical signal and improving sensitivity.26,27 In recent years, various nanomaterials (nanotubes, gold nanoparticles [AuNPs], graphene, etc.) have been applied in different biosensing platforms28,29 to improve already existing biosensing strategies or to develop some new sensing methods.30 Among all the different kinds of nanomaterials, graphene has attracted much attention due to its excellent biocompatibility, large theoretical specic surface area and good electronic property.31,32 However, irreversible agglomeration and poor water solubility limit its further application.33,34 To overcome these shortcomings and introduce more functional groups, tremendous attention has been focused on achieving chemical functionalization of graphene.35 It has been reported that graphene reduced by dopamine (PDA-rGO) has stronger mechanical properties, higher thermal stability and electrical conductivity with the ability to immobilize DNA via the Michael addition reaction.36 Moreover, it also has been reported that PDA-rGO as a biosensor platform has an enhanced surface area for capturing a large number of nanoparticles,37 which provides a platform for signal amplication in electrical biosensing. Herein, we propose a sensitive “signal-on” strategy that depends on cleavage-based HCR for the detection of thrombin (TB). The key conception of this aptasensor was takes advantage of the predominance of TB combination with the thrombin aptamer binding sequence to block the cleavage site of DNAzymes,9 which would prevent the cleavage of DNA by Cu2+dependent DNAzyme and le on the electrode surface. Then, the signal reporter sequence in the binding DNA would act as an initiator to trigger the HCR and to form the dsDNA polymers. The electrochemical signal originated from [Ru(NH3)6]3+, which intercalated into the dsDNA polymers through electrostatic interactions. With the application of PDA-rGO to increase the amount of aptamer chain and promote electron transfer, the linearity between the logarithm of TB concentration and current response provided the quantitative foundation for TB detection with a low detection limit of 0.05 pM.
Experimental
Analyst
auxiliary DNA 1 (A1): 50 –NH2–ACG AAA GAT AGC CAC TCG TAT TCA TCA CTG GAC CGA TAC GCG ACA TAT CGT GCC AAT TAG-30 . Auxiliary DNA 2 (A2): 50 –NH2–TGA CAT TTG CTC GAT TCC TAT ACG AGT GGC TAT CTT TCG TCT AAT TGG CAC GAT ATG TCG-30 . Binding DNA: 50 –NH2–(CH2)6-GAA TTT CTA ATA CGT GGT AGG GCA GGT TGG GGT TAT TAG CTT GAC ATT TGC TCG ATT CCT ATA CGA GTG GCT ATC TTT CGT CTA ATT GGC ACG ATA TGT CG-30 . DNAzyme: 50 -CTACCACTGGGCCTCTTTTTTAAAGAAC-30 . (The binding strength of the TB to the thrombin aptamer binding sequence was designed to be more than the binding strength of the DNAzyme to the thrombin aptamer binding sequence). The aptamer and thrombin solutions were prepared with 20 mM Tris–HCl buffer (pH 7.4) containing 140 mM NaCl, 5 mM KCl and 1 mM MgCl2. 0.1 M phosphate buffered solutions (PBS, pH 7.0) containing 10 mM Na2HPO4, 10 mM KH2PO4 and 2 mM MgCl2 were used throughout the experiment. The human serum samples were obtained from the Ninth People's Hospital of Chongqing, China. Double distilled water was used throughout this study. All the other chemicals were of reagent grade and used as received. Apparatus Cyclic voltammetry (CV) and differential pulse voltammograms (DPV) were performed on a CHI 660D electrochemical workstation (Shanghai Chenhua Instrument, China). All the experiments were performed in a conventional three-electrode system using a platinum wire as the auxiliary electrode, a modied glassy carbon electrode (GCE, F ¼ 4 mm) as the working electrode, and a saturated calomel electrode (SCE) as the reference electrode. CVs of the electrode fabrication were performed in 2 mL of 5 mM K3Fe(CN)6 solution containing 0.2 M KCl with a scanning potential from 0.2 to 0.6 V at a scan rate of 100 mV s1. DPV was performed in 1 mL of 0.1 M PBS (pH 7.0) to measure the amperometric responses of the aptasensor. The parameters applied were: 50 mV pulse amplitude, 50 ms pulse width, 0.2 s pulse period and voltage range from 0.5 to 0 V (vs. SCE). The scanning electron micrographs were obtained on a scanning electron microscope (SEM, S-4800, Hitachi, Japan).
Chemicals and materials
Synthesis of AuNPs
Graphene oxide (GO) was obtained from Nanjing Xian Feng Nano Co. (Nanjing, China). Dopamine hydrochloride (DA) was purchased from Aladdin (Shanghai, China). Tris-hydroxymethylaminomethane hydrochloride (Tris) was obtained from Roche (Switzerland). Thrombin (TB), hexanethiol (96%, HT), gold chloride (1%, HAuCl4), bovine serum albumin (BSA), hexaammineruthenium (III) chloride (99%, [Ru(NH3)6]3+) and hemoglobin (Hb) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). L-Cysteine (L-Cys) and L-ascorbic acid (AA) were obtained from Chengdu Kelong Chemical Industry. All the oligonucleotides were synthesized by Sangon Biotech. Co. Ltd. (Shanghai, China) with the following sequences:
Gold nanoparticles (AuNPs) were prepared by citrate reduction of HAuCl4 according to the classic procedure with certain modications.38 First, 100 mL of 0.01% HAuCl4 solution was prepared. Then, 2.5 mL of 1% trisodium citrate was added under vigorous stirring and boiled for 10 min. Finally, aer stirring for another 20 min, the solution was cooled to room temperature and stored at 4 C.
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Preparation of PDA-rGO composites PDA-rGO composites were prepared according to a method reported in the literature with a slight modication.36 First, 10 mg of GO and 5 mg of dopamine hydrochloride were added
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to 20 mL of 10 mM Tris–HCl solution (pH 8.5), and then dispersed by sonication for 50 min in an ice bath. Subsequently, the reaction mixture was stirred vigorously at 60 C for 24 h and slowly cooled to room temperature. Finally, aer the mixture was centrifuged and washed with double distilled water for three times, the obtained composites were redispersed in PBS (pH 7.0). Fig. 1
SEM micrographs of PDA-rGO (A) and PDA-rGO/nano-Au (B).
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Fabrication of the aptasensor To obtain better results, A1 (2.5 mM) was added to the AuNP solution and incubated for 12 h at 4 C. Prior to use, the GCE was polished with 0.3 and 0.05 mm alumina slurry sequentially and sonicated sequentially with ethanol and double distilled water to obtain a mirror-like surface. First, PDA-rGO was immobilized on the cleaned electrode and dried at room temperature. For the formation of a nano-Au layer on the surface of PDA-rGO, the modied electrode was electrodeposited in HAuCl4 (w/v, 1%) solution at a potential of 0.2 V for 30 s. Then, 20 mL of the binding DNA (2 mM) solution was dropped onto the modied electrode surface for 16 h. Aer the addition of 20 mL of 1.0 mM HT for 50 min to block the remaining active sites, the modied electrode was incubated with 20 mL target TB for 35 min and cleaned. Subsequently, the aptasensor was incubated with 20 mL of a mixture solution including DNAzyme (2 mM), CuCl2 (6 mM) and AA (10 mM) for 5 h. Aer that, 20 mL of A1-AuNP solution was cast onto the constructed electrode for 1 h. Finally, 20 mL of a mixture solution containing A1 (2.5 mM), A2 (2.5 mM) and [Ru(NH3)6]3+ (2 mM) was dropped onto the modied electrode and incubated for 100 min. Aer that, the signal was monitored by DPV in 1.0 M PBS. Scheme 1 shows a schematic illustration of the fabrication of the aptasensor.
prepared successfully. Aer a layer of nano-Au was deposited, several bright points were dispersed on the thin GO layers (Fig. 1B), which demonstrated that nano-Au was immobilized on the PDA-rGO successfully.
Electrochemical characterization of the aptasensor The interface properties of the surface-modied electrodes were investigated by CV measurements. CVs of different modied electrodes in the presence of 5 mM K3[Fe(CN)6] containing 0.1 M KCl were acquired (Fig. 2A). A couple of quasi-reversible and well-dened redox peaks of [Fe(CN)6]4/3 were observed for the pretreated bare GCE (Fig. 2A, curve a). The current response increased (Fig. 2A, curve b) aer a lm of PDA-rGO was immobilized on the electrode surface, which was attributed to PDA-rGO enhancing the electrical conductivity.36 Because of the strong capture capabilities of PDA-rGO for nano-Au, the peak current markedly increased (Fig. 2A, curve c) and was higher (Fig. 2C, curve b) than the signal of bare GCE straightforwardly
Results and discussion Characteristics of different nanomaterials The sizes and morphologies of the synthesized nanocomposites were characterized by SEM. As shown in Fig. 1A, PDA-rGO exhibited typical wrinkled morphology and was exfoliated into single or a layer of tulle, indicating that the PDA-rGO was
(A) CVs of different electrodes: (a) bare GCE, (b) GCE/PDA-rGO, (c) GCE/PDA-rGO/nano-Au, (d) GCE/PDA-rGO/nano-Au/binding DNA, (e) GCE/PDA-rGO/nano-Au/binding DNA/HT, (f) GCE/PDArGO/nano-Au/binding DNA/HT/TB, (g) GCE/PDA-rGO/nano-Au/ binding DNA/HT/TB/DNAzyme, and (h) GCE/PDA-rGO/nano-Au/ binding DNA/HT/TB/DNAzyme/A1-AuNPs. (B) DPV responses of the resulting aptasensors for TB detection: the aptasensor before (a) and after the induction of HCR (b). (C) A comparison of the current signal for GCE/nano-Au (a) and GCE/PDA-rGO/nano-Au (b). Fig. 2
Scheme 1 Illustration of the stepwise aptasensor fabrication based on Cu2+-dependent DNAzyme as the cleavage enzyme and HCR for amplification.
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modied with a layer of nano-Au (Fig. 2C, curve a), implying that PDA-rGO/nano-Au could more clearly enhance electron transfer efficiency. Aer binding DNA was assembled on the resulting electrode, the electron transfer tunnel was blocked and the peak current decreased (Fig. 2A, curve d). As expected, the peak current decreased (Fig. 2A, curve e) aer employing HT to block nonspecic sites. A further decrease in the peak current was obtained during the binding DNA formation of a G-quartet to bind TB (Fig. 2A, curve f). Subsequently, when DNAzyme was immobilized on the modied electrode, the current increased (Fig. 2A, curve g) because Cu2+-dependent DNAzyme cleaved binding DNA that did not form a protein-aptamer complex, resulting in less DNA on the electrode surface to hinder the electron transfer. Finally, aer the immobilization of A1-AuNPs, the current signal of the resulting aptasensor further increased because AuNPs were able to improve electron transport. Moreover, the electrochemical characteristics of the aptasensor before and aer the induction of HCR were investigated by DPV measurements in 1.0 mL PBS (Fig. 2B). Aer incubation with the mixture of A1-AuNPs, no peak was observed due to the lack of an electroactive probe (Fig. 2B, curve a). A strong peak current of [Ru(NH3)6]3+ appeared (Fig. 2B, curve b) aer HCR was triggered by the signal reporter sequence. These results strongly support the claim of successful fabrication via HCR. The HCR achieved with two helpers, A1 and A2, was further examined by gel electrophoresis (see the ESI†). Optimization of experimental conditions for aptasensor The target incubation time was an important factor for the formation of the aptamer-target structure. Therefore, to increase the sensitivity and selectivity of the aptasensor, we investigated the incubation time of TB in this work. As shown in Fig. 3A, the electrochemical signal of [Fe(CN)6]4/3 intensively decreased with a gradual increase in incubation time and appeared saturated aer incubation for 35 min. Taking this into consideration, 35 min was chosen as the optimal incubation time for TB in this study. The hybridization time for HCR also inuenced the performance of the aptasensor. As shown in Fig. 3B, the DPV signals for [Ru(NH3)6]3+ increased rapidly with an increase in hybridization time and almost leveled off aer 100 min. Consequently, 100 min was chosen as the optimal incubation time for the hybridization of HCR.
Analyst
DPV response and calibration curves In the experiments, the DPV signals of different concentrations of TB were measured in 1.0 mL PBS under optimal conditions. The response currents of DPV increased gradually with an increase in TB concentration (Fig. 4A). The calibration plots showed a linear relationship between the reduction peak currents and the logarithm of TB concentrations from 0.0001 nM to 50 nM (Fig. 4B). The linear regression equation was adjusted to I (mA) ¼ 40.53–8.348 log c TB (nM, r ¼ 0.9922) with a detection limit of 0.05 pM (dened as DL ¼ 3SB/m, where m is the slope of the corresponding calibration curve and SB is the standard deviation of the blank), indicating that TB concentration was quantitatively measured by DPV signal with high sensitivity. In addition, the analytical performance of the developed aptasensor was compared with that of the other similar detection methods for thrombin detection. The results are summarized in Table S1 (see the ESI†).
Selectivity, reproducibility and stability of the aptasensor The specicity of the aptasensor was investigated (Fig. 5). The inuences of interfering agents, such as BSA (100 nM), Hb (100 nM), and L-cys (100 nM), were examined under the same experimental conditions, and a comparative experiment was carried out by measuring the DPV response of the aptasensor in pure 10 nM TB. Even with a high concentration of interfering components (100 nM), no apparent changes in the current were observed alone. However, the current was dramatically
(A) DPVs of the aptasensor with different concentrations of TB: 0 nM, 0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, and 50 nM in 1.0 mL 0.1 M PBS. (B) The calibration curve of current response to the logarithm of TB at different concentrations.
Fig. 4
Selectivity investigation compared with different targets: (a) blank solution (0 nM thrombin), (b) 100 nM BSA, (c) 100 nM Hb, (d) 100 nM L-cys, (f) 10 nM TB + 100 nM BSA+ 100 nM Hb + 100 nM L-cys, and (f) 10 nM TB. Fig. 5
Optimization of experimental parameters: (A) influence of incubation time with TB on the current response of the aptasensor. (B) Effect of hybridization time on HCR amplification. Fig. 3
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Table 1
Paper Determination of TB added to human blood serum (n ¼ 3) with the proposed aptasensor
Sample
Added thrombin/nM
Found thrombin/nM
Recovery/%
RSD (%)
1 2 3 4 5
0.1 1.0 10.0 20.0 30.0
0.1076 0.9160 10.0324 19.9243 30.1107
107.6 91.6 100.3 99.6 100.4
4.9 3.4 5.6 6.5 4.2
increased in the presence of target TB (10 nM), indicating the advantageous specicity of the proposed amplication strategy in this assay. The reproducibility of the proposed aptasensor was investigated according to the intra-assay and inter-assay relative standard deviations (RSDs, n ¼ 5). The signal of [Ru(NH3)6]3+ was detected by different electrodes at the same conditions, and the RSDs with different concentrations of 0.1 nM and 20 nM were 5.1% and 4.6%, respectively. In addition, the reproducibility was also assessed via measuring in duplicate the TB level with ve different electrodes, and the RSDs with 0.01 nM and 30 nM were 4.8% and 4.3%, respectively. These results demonstrate that the reproducibility of the proposed aptasensor was acceptable. In this work, the long-term storage stability of the proposed aptasensor was studied over a 20 day period. In this period, the aptasensor was stored in a refrigerator at 4 C and tested every 5 days. The current signal of the DPV decreased gradually by about 4.5% of its initial current aer 5 days, and 91.2% of the initial current was retained aer 20 days, suggesting that the assembled aptasensor had sufficient stability for the detection of TB. Preliminary analysis of real samples To further demonstrate the merits of the prepared aptasensor for the detection of TB, ve samples containing 0.1 nM, 1 nM, 10 nM, 20.0 nM and 30.0 nM target TB were obtained by spiking 10-fold-diluted human real serum samples with TB standards. As shown in Table 1, the recovery and RSDs ranged from 91.6% to 107.6% and 3.4–6.5%, respectively, which conrmed that this aptasensor shows potential for TB detection in real biological samples.
Conclusions In summary, we constructed a novel aptasensor to detect TB with good specicity, acceptable reproducibility and stability. The high sensitivity of the aptasensor may be due to the following reasons: (1) a new “signal-on” strategy was constructed based on the formation of aptamer-protein complexation and the use of a new kind of Cu2+-dependent DNAzyme cleavage enzyme; (2) the dsDNA polymers formed by HCR could capture a large amount of electron mediator [Ru(NH3)6]3+ to produce a high electrochemical signal; (3) and the use of functionalized PDA-rGO could further enhance the current signal and improve the sensitivity of this aptasensor. Based on
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these advantages, we anticipate that the proposed method may have excellent potential applications in clinical diagnosis.
Acknowledgements This work was nancially supported by the NNSF of China (21275119, 21075100), State Key Laboratory of Electroanalytical Chemistry (SKLEAC2010009), Ministry of Education of China (Project 708073), Natural Science Foundation of Chongqing City (CSTC-2010BB4121, CSTC-2011BA7003), and Specialized Research Fund for the Doctoral Program of Higher Education (20100182110015).
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Cleavage-based hybridization chain reaction for electrochemical detection of thrombin† Yuanyuan Chang, Yaqin Chai,* Shunbi Xie, Yali Yuan, Juan Zhang and Ruo Yuan* In the present work, we constructed a new label-free “inter-sandwich” electrochemical aptasensor for thrombin (TB) detection by employing a cleavage-based hybridization chain reaction (HCR). The designed single-stranded DNA (defined as binding DNA), which contained the thrombin aptamer binding sequence, a DNAzyme cleavage site and a signal reporter sequence, was first immobilized on the electrode. In the absence of a target TB, the designed DNAzymes could combine with the thrombin aptamer binding sequence via complementary base pairing, and then Cu2+ could cleave the binding DNA. In the presence of a target TB, TB could combine with the thrombin aptamer binding sequence to predominantly form an aptamer–protein complex, which blocked the DNAzyme cleavage site and prevented the binding DNA from being cleaved by Cu2+-dependent DNAzyme. As a result, the signal reporter sequence could leave the electrode surface to trigger HCR with the help of two auxiliary DNA single-strands, A1 and A2. Then, the electron mediator hexaammineruthenium (III) chloride ([Ru(NH3)6]3+) was embedded into the double-stranded DNA (dsDNA) to produce a strong electrochemical signal for the quantitative measurement of TB. For further amplification of the electrochemical signal, graphene reduced by dopamine (PDA-rGO) was introduced as a platform in this work. With this strategy, the
Received 22nd April 2014 Accepted 6th June 2014
aptasensor displayed a wide linearity in the range of 0.0001 nM to 50 nM with a low detection limit of 0.05 pM. Moreover, the resulting aptasensor exhibited good specificity and acceptable reproducibility
DOI: 10.1039/c4an00712c
and stability. Because of these factors, the fabrication protocol proposed in this work may be extended to clinical application.
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Introduction The functional nucleic acid DNAzymes (also known as catalytic DNA, deoxyribozymes, or DNA enzymes) are a kind of articial enzymes.1,2 In addition their function as catalysts, another attractive aspect of DNAzymes is that they have a metal-iondependent cleavage specic, partially complementary DNA substrate in the presence of metal ions.3 The special cleavage enzyme possesses the qualities of being relatively less expensive, easily labeled, highly stable and offering straightforward sequence specic recognition.4–6 Because of these properties, metal-ion-dependent DNAzymes, as cleavage enzymes, have exhibited surprising potential in biochemical reactions in recent years. For example, Gao's group successfully constructed a lead biosensor based on GR-5 lead-dependent DNAzyme.7 Zhang's group proposed a new electrochemical immunoassay protocol based on the cleavage of metal-ion-induced DNAzymes for the determination of protein with high sensitivity and Education Ministry Key Laboratory of Luminescent and Real-Time Analytical Chemistry, College of Chemistry and Chemical Engineering, Chongqing 400715, People's Republic of China. E-mail: [email protected]; [email protected]; Fax: +86-23-68253172; Tel: +86-23-68252277 † Electronic supplementary 10.1039/c4an00712c
information
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(ESI)
available.
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DOI:
specicity.8 Furthermore, Chen's group recently reported a novel method that chose Cu2+-dependent. DNAzymes as the cleavage enzyme to quantitatively measure protein-aptamer complexation on the surface by blocking DNAzyme cleavage activity.9 Therefore, metal-ion-dependent DNAzymes as a novel kind of cleavage enzymes provide a new path to develop various biosensors. The improvement of electrochemical biosensors with high sensitivity aims at reducing detection limits and amplifying the signal by using various methods.10 Thus, numerous DNA amplication technologies, including polymerase chain reaction (PCR),11 rolling circle amplication (RCA),12 strand displacement amplication (SDA)13 and hybridization chain reaction (HCR)14–16 have been developed. Among these methods, HCR is one of the most frequently used strategies, especially in the eld of electrochemical technology.17–20 HCR is a self-assembled and enzyme-free process that can perform a cascade of hybridization events with two single-stranded DNA (ssDNA) molecules to spontaneously form a long doublestranded DNA (dsDNA) molecule based on the energy of base pair formation under mild conditions14,21–23 in the presence of an initiator DNA. The formed dsDNA with a negative charge and stably rigid structure was an ideal carrier for loading numerous electrochemically active compounds via electrostatic adsorption
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or labeling.24 For instance, the positively charged electron mediator hexaammineruthenium (III) chloride ([Ru(NH3)6]3+) can automatically embed into dsDNA via electrostatic adsorption,25 and the process avoids the complex labeling steps by enhancing electrochemical signal and improving sensitivity.26,27 In recent years, various nanomaterials (nanotubes, gold nanoparticles [AuNPs], graphene, etc.) have been applied in different biosensing platforms28,29 to improve already existing biosensing strategies or to develop some new sensing methods.30 Among all the different kinds of nanomaterials, graphene has attracted much attention due to its excellent biocompatibility, large theoretical specic surface area and good electronic property.31,32 However, irreversible agglomeration and poor water solubility limit its further application.33,34 To overcome these shortcomings and introduce more functional groups, tremendous attention has been focused on achieving chemical functionalization of graphene.35 It has been reported that graphene reduced by dopamine (PDA-rGO) has stronger mechanical properties, higher thermal stability and electrical conductivity with the ability to immobilize DNA via the Michael addition reaction.36 Moreover, it also has been reported that PDA-rGO as a biosensor platform has an enhanced surface area for capturing a large number of nanoparticles,37 which provides a platform for signal amplication in electrical biosensing. Herein, we propose a sensitive “signal-on” strategy that depends on cleavage-based HCR for the detection of thrombin (TB). The key conception of this aptasensor was takes advantage of the predominance of TB combination with the thrombin aptamer binding sequence to block the cleavage site of DNAzymes,9 which would prevent the cleavage of DNA by Cu2+dependent DNAzyme and le on the electrode surface. Then, the signal reporter sequence in the binding DNA would act as an initiator to trigger the HCR and to form the dsDNA polymers. The electrochemical signal originated from [Ru(NH3)6]3+, which intercalated into the dsDNA polymers through electrostatic interactions. With the application of PDA-rGO to increase the amount of aptamer chain and promote electron transfer, the linearity between the logarithm of TB concentration and current response provided the quantitative foundation for TB detection with a low detection limit of 0.05 pM.
Experimental
Analyst
auxiliary DNA 1 (A1): 50 –NH2–ACG AAA GAT AGC CAC TCG TAT TCA TCA CTG GAC CGA TAC GCG ACA TAT CGT GCC AAT TAG-30 . Auxiliary DNA 2 (A2): 50 –NH2–TGA CAT TTG CTC GAT TCC TAT ACG AGT GGC TAT CTT TCG TCT AAT TGG CAC GAT ATG TCG-30 . Binding DNA: 50 –NH2–(CH2)6-GAA TTT CTA ATA CGT GGT AGG GCA GGT TGG GGT TAT TAG CTT GAC ATT TGC TCG ATT CCT ATA CGA GTG GCT ATC TTT CGT CTA ATT GGC ACG ATA TGT CG-30 . DNAzyme: 50 -CTACCACTGGGCCTCTTTTTTAAAGAAC-30 . (The binding strength of the TB to the thrombin aptamer binding sequence was designed to be more than the binding strength of the DNAzyme to the thrombin aptamer binding sequence). The aptamer and thrombin solutions were prepared with 20 mM Tris–HCl buffer (pH 7.4) containing 140 mM NaCl, 5 mM KCl and 1 mM MgCl2. 0.1 M phosphate buffered solutions (PBS, pH 7.0) containing 10 mM Na2HPO4, 10 mM KH2PO4 and 2 mM MgCl2 were used throughout the experiment. The human serum samples were obtained from the Ninth People's Hospital of Chongqing, China. Double distilled water was used throughout this study. All the other chemicals were of reagent grade and used as received. Apparatus Cyclic voltammetry (CV) and differential pulse voltammograms (DPV) were performed on a CHI 660D electrochemical workstation (Shanghai Chenhua Instrument, China). All the experiments were performed in a conventional three-electrode system using a platinum wire as the auxiliary electrode, a modied glassy carbon electrode (GCE, F ¼ 4 mm) as the working electrode, and a saturated calomel electrode (SCE) as the reference electrode. CVs of the electrode fabrication were performed in 2 mL of 5 mM K3Fe(CN)6 solution containing 0.2 M KCl with a scanning potential from 0.2 to 0.6 V at a scan rate of 100 mV s1. DPV was performed in 1 mL of 0.1 M PBS (pH 7.0) to measure the amperometric responses of the aptasensor. The parameters applied were: 50 mV pulse amplitude, 50 ms pulse width, 0.2 s pulse period and voltage range from 0.5 to 0 V (vs. SCE). The scanning electron micrographs were obtained on a scanning electron microscope (SEM, S-4800, Hitachi, Japan).
Chemicals and materials
Synthesis of AuNPs
Graphene oxide (GO) was obtained from Nanjing Xian Feng Nano Co. (Nanjing, China). Dopamine hydrochloride (DA) was purchased from Aladdin (Shanghai, China). Tris-hydroxymethylaminomethane hydrochloride (Tris) was obtained from Roche (Switzerland). Thrombin (TB), hexanethiol (96%, HT), gold chloride (1%, HAuCl4), bovine serum albumin (BSA), hexaammineruthenium (III) chloride (99%, [Ru(NH3)6]3+) and hemoglobin (Hb) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). L-Cysteine (L-Cys) and L-ascorbic acid (AA) were obtained from Chengdu Kelong Chemical Industry. All the oligonucleotides were synthesized by Sangon Biotech. Co. Ltd. (Shanghai, China) with the following sequences:
Gold nanoparticles (AuNPs) were prepared by citrate reduction of HAuCl4 according to the classic procedure with certain modications.38 First, 100 mL of 0.01% HAuCl4 solution was prepared. Then, 2.5 mL of 1% trisodium citrate was added under vigorous stirring and boiled for 10 min. Finally, aer stirring for another 20 min, the solution was cooled to room temperature and stored at 4 C.
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Preparation of PDA-rGO composites PDA-rGO composites were prepared according to a method reported in the literature with a slight modication.36 First, 10 mg of GO and 5 mg of dopamine hydrochloride were added
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to 20 mL of 10 mM Tris–HCl solution (pH 8.5), and then dispersed by sonication for 50 min in an ice bath. Subsequently, the reaction mixture was stirred vigorously at 60 C for 24 h and slowly cooled to room temperature. Finally, aer the mixture was centrifuged and washed with double distilled water for three times, the obtained composites were redispersed in PBS (pH 7.0). Fig. 1
SEM micrographs of PDA-rGO (A) and PDA-rGO/nano-Au (B).
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Fabrication of the aptasensor To obtain better results, A1 (2.5 mM) was added to the AuNP solution and incubated for 12 h at 4 C. Prior to use, the GCE was polished with 0.3 and 0.05 mm alumina slurry sequentially and sonicated sequentially with ethanol and double distilled water to obtain a mirror-like surface. First, PDA-rGO was immobilized on the cleaned electrode and dried at room temperature. For the formation of a nano-Au layer on the surface of PDA-rGO, the modied electrode was electrodeposited in HAuCl4 (w/v, 1%) solution at a potential of 0.2 V for 30 s. Then, 20 mL of the binding DNA (2 mM) solution was dropped onto the modied electrode surface for 16 h. Aer the addition of 20 mL of 1.0 mM HT for 50 min to block the remaining active sites, the modied electrode was incubated with 20 mL target TB for 35 min and cleaned. Subsequently, the aptasensor was incubated with 20 mL of a mixture solution including DNAzyme (2 mM), CuCl2 (6 mM) and AA (10 mM) for 5 h. Aer that, 20 mL of A1-AuNP solution was cast onto the constructed electrode for 1 h. Finally, 20 mL of a mixture solution containing A1 (2.5 mM), A2 (2.5 mM) and [Ru(NH3)6]3+ (2 mM) was dropped onto the modied electrode and incubated for 100 min. Aer that, the signal was monitored by DPV in 1.0 M PBS. Scheme 1 shows a schematic illustration of the fabrication of the aptasensor.
prepared successfully. Aer a layer of nano-Au was deposited, several bright points were dispersed on the thin GO layers (Fig. 1B), which demonstrated that nano-Au was immobilized on the PDA-rGO successfully.
Electrochemical characterization of the aptasensor The interface properties of the surface-modied electrodes were investigated by CV measurements. CVs of different modied electrodes in the presence of 5 mM K3[Fe(CN)6] containing 0.1 M KCl were acquired (Fig. 2A). A couple of quasi-reversible and well-dened redox peaks of [Fe(CN)6]4/3 were observed for the pretreated bare GCE (Fig. 2A, curve a). The current response increased (Fig. 2A, curve b) aer a lm of PDA-rGO was immobilized on the electrode surface, which was attributed to PDA-rGO enhancing the electrical conductivity.36 Because of the strong capture capabilities of PDA-rGO for nano-Au, the peak current markedly increased (Fig. 2A, curve c) and was higher (Fig. 2C, curve b) than the signal of bare GCE straightforwardly
Results and discussion Characteristics of different nanomaterials The sizes and morphologies of the synthesized nanocomposites were characterized by SEM. As shown in Fig. 1A, PDA-rGO exhibited typical wrinkled morphology and was exfoliated into single or a layer of tulle, indicating that the PDA-rGO was
(A) CVs of different electrodes: (a) bare GCE, (b) GCE/PDA-rGO, (c) GCE/PDA-rGO/nano-Au, (d) GCE/PDA-rGO/nano-Au/binding DNA, (e) GCE/PDA-rGO/nano-Au/binding DNA/HT, (f) GCE/PDArGO/nano-Au/binding DNA/HT/TB, (g) GCE/PDA-rGO/nano-Au/ binding DNA/HT/TB/DNAzyme, and (h) GCE/PDA-rGO/nano-Au/ binding DNA/HT/TB/DNAzyme/A1-AuNPs. (B) DPV responses of the resulting aptasensors for TB detection: the aptasensor before (a) and after the induction of HCR (b). (C) A comparison of the current signal for GCE/nano-Au (a) and GCE/PDA-rGO/nano-Au (b). Fig. 2
Scheme 1 Illustration of the stepwise aptasensor fabrication based on Cu2+-dependent DNAzyme as the cleavage enzyme and HCR for amplification.
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modied with a layer of nano-Au (Fig. 2C, curve a), implying that PDA-rGO/nano-Au could more clearly enhance electron transfer efficiency. Aer binding DNA was assembled on the resulting electrode, the electron transfer tunnel was blocked and the peak current decreased (Fig. 2A, curve d). As expected, the peak current decreased (Fig. 2A, curve e) aer employing HT to block nonspecic sites. A further decrease in the peak current was obtained during the binding DNA formation of a G-quartet to bind TB (Fig. 2A, curve f). Subsequently, when DNAzyme was immobilized on the modied electrode, the current increased (Fig. 2A, curve g) because Cu2+-dependent DNAzyme cleaved binding DNA that did not form a protein-aptamer complex, resulting in less DNA on the electrode surface to hinder the electron transfer. Finally, aer the immobilization of A1-AuNPs, the current signal of the resulting aptasensor further increased because AuNPs were able to improve electron transport. Moreover, the electrochemical characteristics of the aptasensor before and aer the induction of HCR were investigated by DPV measurements in 1.0 mL PBS (Fig. 2B). Aer incubation with the mixture of A1-AuNPs, no peak was observed due to the lack of an electroactive probe (Fig. 2B, curve a). A strong peak current of [Ru(NH3)6]3+ appeared (Fig. 2B, curve b) aer HCR was triggered by the signal reporter sequence. These results strongly support the claim of successful fabrication via HCR. The HCR achieved with two helpers, A1 and A2, was further examined by gel electrophoresis (see the ESI†). Optimization of experimental conditions for aptasensor The target incubation time was an important factor for the formation of the aptamer-target structure. Therefore, to increase the sensitivity and selectivity of the aptasensor, we investigated the incubation time of TB in this work. As shown in Fig. 3A, the electrochemical signal of [Fe(CN)6]4/3 intensively decreased with a gradual increase in incubation time and appeared saturated aer incubation for 35 min. Taking this into consideration, 35 min was chosen as the optimal incubation time for TB in this study. The hybridization time for HCR also inuenced the performance of the aptasensor. As shown in Fig. 3B, the DPV signals for [Ru(NH3)6]3+ increased rapidly with an increase in hybridization time and almost leveled off aer 100 min. Consequently, 100 min was chosen as the optimal incubation time for the hybridization of HCR.
Analyst
DPV response and calibration curves In the experiments, the DPV signals of different concentrations of TB were measured in 1.0 mL PBS under optimal conditions. The response currents of DPV increased gradually with an increase in TB concentration (Fig. 4A). The calibration plots showed a linear relationship between the reduction peak currents and the logarithm of TB concentrations from 0.0001 nM to 50 nM (Fig. 4B). The linear regression equation was adjusted to I (mA) ¼ 40.53–8.348 log c TB (nM, r ¼ 0.9922) with a detection limit of 0.05 pM (dened as DL ¼ 3SB/m, where m is the slope of the corresponding calibration curve and SB is the standard deviation of the blank), indicating that TB concentration was quantitatively measured by DPV signal with high sensitivity. In addition, the analytical performance of the developed aptasensor was compared with that of the other similar detection methods for thrombin detection. The results are summarized in Table S1 (see the ESI†).
Selectivity, reproducibility and stability of the aptasensor The specicity of the aptasensor was investigated (Fig. 5). The inuences of interfering agents, such as BSA (100 nM), Hb (100 nM), and L-cys (100 nM), were examined under the same experimental conditions, and a comparative experiment was carried out by measuring the DPV response of the aptasensor in pure 10 nM TB. Even with a high concentration of interfering components (100 nM), no apparent changes in the current were observed alone. However, the current was dramatically
(A) DPVs of the aptasensor with different concentrations of TB: 0 nM, 0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, and 50 nM in 1.0 mL 0.1 M PBS. (B) The calibration curve of current response to the logarithm of TB at different concentrations.
Fig. 4
Selectivity investigation compared with different targets: (a) blank solution (0 nM thrombin), (b) 100 nM BSA, (c) 100 nM Hb, (d) 100 nM L-cys, (f) 10 nM TB + 100 nM BSA+ 100 nM Hb + 100 nM L-cys, and (f) 10 nM TB. Fig. 5
Optimization of experimental parameters: (A) influence of incubation time with TB on the current response of the aptasensor. (B) Effect of hybridization time on HCR amplification. Fig. 3
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Table 1
Paper Determination of TB added to human blood serum (n ¼ 3) with the proposed aptasensor
Sample
Added thrombin/nM
Found thrombin/nM
Recovery/%
RSD (%)
1 2 3 4 5
0.1 1.0 10.0 20.0 30.0
0.1076 0.9160 10.0324 19.9243 30.1107
107.6 91.6 100.3 99.6 100.4
4.9 3.4 5.6 6.5 4.2
increased in the presence of target TB (10 nM), indicating the advantageous specicity of the proposed amplication strategy in this assay. The reproducibility of the proposed aptasensor was investigated according to the intra-assay and inter-assay relative standard deviations (RSDs, n ¼ 5). The signal of [Ru(NH3)6]3+ was detected by different electrodes at the same conditions, and the RSDs with different concentrations of 0.1 nM and 20 nM were 5.1% and 4.6%, respectively. In addition, the reproducibility was also assessed via measuring in duplicate the TB level with ve different electrodes, and the RSDs with 0.01 nM and 30 nM were 4.8% and 4.3%, respectively. These results demonstrate that the reproducibility of the proposed aptasensor was acceptable. In this work, the long-term storage stability of the proposed aptasensor was studied over a 20 day period. In this period, the aptasensor was stored in a refrigerator at 4 C and tested every 5 days. The current signal of the DPV decreased gradually by about 4.5% of its initial current aer 5 days, and 91.2% of the initial current was retained aer 20 days, suggesting that the assembled aptasensor had sufficient stability for the detection of TB. Preliminary analysis of real samples To further demonstrate the merits of the prepared aptasensor for the detection of TB, ve samples containing 0.1 nM, 1 nM, 10 nM, 20.0 nM and 30.0 nM target TB were obtained by spiking 10-fold-diluted human real serum samples with TB standards. As shown in Table 1, the recovery and RSDs ranged from 91.6% to 107.6% and 3.4–6.5%, respectively, which conrmed that this aptasensor shows potential for TB detection in real biological samples.
Conclusions In summary, we constructed a novel aptasensor to detect TB with good specicity, acceptable reproducibility and stability. The high sensitivity of the aptasensor may be due to the following reasons: (1) a new “signal-on” strategy was constructed based on the formation of aptamer-protein complexation and the use of a new kind of Cu2+-dependent DNAzyme cleavage enzyme; (2) the dsDNA polymers formed by HCR could capture a large amount of electron mediator [Ru(NH3)6]3+ to produce a high electrochemical signal; (3) and the use of functionalized PDA-rGO could further enhance the current signal and improve the sensitivity of this aptasensor. Based on
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these advantages, we anticipate that the proposed method may have excellent potential applications in clinical diagnosis.
Acknowledgements This work was nancially supported by the NNSF of China (21275119, 21075100), State Key Laboratory of Electroanalytical Chemistry (SKLEAC2010009), Ministry of Education of China (Project 708073), Natural Science Foundation of Chongqing City (CSTC-2010BB4121, CSTC-2011BA7003), and Specialized Research Fund for the Doctoral Program of Higher Education (20100182110015).
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