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Target-induced reconfiguration of DNA probes for recycling amplification and signal-on electrochemical detection of hereditary tyrosinemia type I gene Baoting Dou, Cuiyun Yang, Yaqin Chai, Ruo Yuan and Yun Xiang* By coupling target DNA-induced reconfiguration of the dsDNA probes with enzyme-assisted target recycling amplification, we describe the development of a signal-on electrochemical sensing approach for sensitive detection of hereditary tyrosinemia type I gene. The dsDNA probes are self-assembled on the sensing electrode, and the addition of the target DNA reconfigures and switches the dsDNA probes into active substrates for exonuclease III, which catalytically digests the probes and leads to cyclic reuse of the target DNA. The target DNA recycling and the removal of one of the ssDNA from the dsDNA probes by exonuclease III result in the formation of many hairpin structures on the sensor surface, which brings the electroactive methylene blue labels into proximity with the electrode and produces a significantly amplified
Received 19th May 2015, Accepted 26th June 2015
current response for sensitive detection of the target gene down to 0.24 pM. This method is also selective
DOI: 10.1039/c5an01006c
to discriminate single-base mismatch and can be employed to detect the target gene in human serum samples. With the demonstration for the detection of the target gene, we expect the developed method to
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be a universal sensitive sensing platform for the detection of different nucleic acid sequences.
Introduction The identification and detection of sequence specific DNA plays important roles in molecular diagnostics,1,2 forensic investigations,3 genetics therapy,4 and biomedical 5–8 development. Achieving high sensitivity and low detection limit is one of the major goals in developing different DNA sensing methods, and a variety of signal amplifications have been demonstrated to meet this goal. Traditional signal amplification methods including polymerase chain reaction (PCR)9 and rolling circle amplification (RCA)10,11 and so on can realize sensitive detection of DNA but are accompanied by the drawbacks of sophisticated instrumentation, easy contamination and high cost. This has led to the development of new signal amplification strategies for detecting low levels of DNA. The post-amplification strategies with the signals produced by hybridization events and the newly developed target cycling strategies have become the most common signal amplification platforms for the detection of DNA. The former relies on sequence specific hybridization recognition of the target DNA and the use of amplification labels such as enzymes,12,13 nano-
Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China. E-mail: [email protected]; Fax: +86-23-68252277; Tel: +86-23-68253172
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materials,14,15 DNA concatamers,16,17 and DNA biobarcodes18 to generate an amplified signal output. These amplification labels usually involve multiple assay steps and require the addition of many exogenous reagents. For example, nanomaterials provide the opportunity for amplifying hybridization signals of DNA biosensors by incorporating DNA with nanomaterials, due to their unique chemical and physical properties caused by their quantum confinement, surface, small size, and macro-quantum tunnel effects. However, these nanomaterials require complex fabrication processes and highly trained technicians.19–21 The target recycling amplification strategies, which involve the cyclic usage of the target molecules, have recently gained increased attention in DNA sensing. The achievement of target DNA recycling in these approaches is based on catalytic digestion of the hybridizedprobe sequences or displacement of the target DNA in the presence of a primer and polymerase upon the hybridizations between the target and probe sequences with the assistance of various nucleases. Without the requirement of a designated primer, the target recycling by nuclease catalytic digestion, including endonucleases (restriction enzymes)22–24 and exonucleases,25,26 can also achieve greatly enhanced sensitivity owing to the multiple hybridization events of the target sequences. Both endonucleases and exonucleases can digest the oligonucleotide probes hybridized with the target DNA selectively and release the target DNA to achieve target DNA
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recycling. Considering the fact that specific nucleotide sequences must be included in the oligonucleotide probes when using the endonucleases,22,27 the use of exonucleases for target DNA recycling without the requirement of specific sequences in the probes is more convenient, and a large number of target DNA recycling methods with the assistance of exonucleases have been developed to achieve sensitive detection of DNA in connection with fluorescent,28,29 electrochemical,30–32 surface plasmon resonance (SPR)33 or surface enhanced resonance Raman scattering (SERS)34,35 transduction means. Among these amplified detection schemes, the electrochemical approaches have received particular interest because of the intrinsic advantages of these techniques in terms of simplicity, low cost, portability and high sensitivity. Herein, based on a new target-induced reconfiguration strategy, we report on an enzyme-assisted target recycling amplification approach for sensitive electrochemical detection of hereditary tyrosinemia type I gene, a biomarker for the fumarylacetoacetate hydrolase gene mutation.36,37 The target DNAs hybridize with the dsDNA probes on the electrode surface and reconfigure the probes into active substrates for exonuclease, which cleaves one of the ssDNA strands in the DNA duplex probes and recycles the target DNA, generating amplified and “turn-on” electrochemical signals for detecting the hereditary tyrosinemia type I gene down to the sub-picomolar level.
Experimental Materials and reagents Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and 6-mercapto-1-hexanol (MCH) were obtained from SigmaAldrich (St. Louis, MO, USA). Exonuclease III (Exo III) and 10× NEBuffer 1 (1× NEBuffer 1, 10 mM bis-tris-propane-HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.0) were purchased from New England Biolabs (Beijing, China). The HPLC-purified oligonucleotides with sequences listed below were ordered from Sangon Biotech. Co., Ltd (Shanghai, China). Thiolated and methylene blue (MB)-modified hairpin DNA signal probe (SP): 5′-MB-(CH2)7-GAC GCT AAT AAT AGC ATA GCG TC-(CH2)6-SH-3′; assistant probe (AP): 5′-TGC TAT TAT TAG CGT CAT TCA CCG G-3′; target DNA (target): 5′-CCG GTG AAT ATC TGG-3′; single-base mismatched DNA (sDNA): 5′-CCG GC̲G AAT ATC TGG-3′; non-complementary DNA (nDNA): 5′TAT TCA GCG GCA CTA-3′. The bold and italic sequences, respectively, correspond to the complementary sequences between SP/AP and AP/target. All other chemicals used were of analytical reagent grade and all aqueous solutions were prepared with deionized water (specific resistance of 18 MΩ cm). Pretreatment of the gold electrode Prior to use, the 3.0 mm diameter gold electrodes (AuEs, CH Instruments Inc., Shanghai, China) were soaked in a freshly
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prepared Piranha solution (mixture of 98% H2SO4 and 30% H2O2 at a volume ratio of 3 : 1) for 0.5 hour. After rinsing thoroughly with water, the AuEs were polished with 0.3 and 0.05 µm aluminum slurry for 5 min separately, followed by sonicating in pure water, ethanol and pure water for 5 minutes, respectively, to remove the residual alumina powder. Next, each AuE was electrochemically cleaned in 0.5 M H2SO4 with the potential scanning from 0.3 to 1.5 V until stable characteristic voltammetric peaks were obtained. Following being dried with nitrogen, the cleaned electrodes were immediately used for probe immobilization. Sensor fabrication Firstly, the dsDNA probes (SP–AP) were obtained by annealing the mixture of SP (2.0 µM) and AP (2.2 µM) in 10 mM phosphate buffer solution (0.1 M NaCl, pH 7.4) at 85 °C for 10 minutes, followed by cooling down to 25 °C in 1 h. Subsequently, the annealed dsDNA probes (0.2 µM) were incubated with TCEP (10 mM) for 1 hour to reduce the disulfide bond and the MB moiety of SP. Next, droplets of 10 µL of the reduced probes were cast on the pretreated AuE surface and incubated overnight. After that, the electrodes were rinsed with water and then blocked with 1 mM MCH for 2 h to prepare the MCH/dsDNA/AuE modified electrodes. The modified electrodes were then immersed in 20 mM Tris buffer (140 mM NaCl, 5 mM MgCl2, pH 7.4) and allowed to equilibrate for 1 h, followed by measuring the responses of MB by square wave voltammetry (SWV) in 20 mM Tris buffer (140 mM NaCl, 5 mM MgCl2, pH 7.4) every fifteen minutes until the variation in MB signals was less than 1%. After getting the background current, the modified electrodes were incubated with 10 µL of solution containing various concentrations of target DNA and 10 U Exo III at 37 °C for 120 min. The sensors were further measured by SWV to obtain the signal responses. Electrochemical measurements Cyclic voltammetry (CV) and square wave voltammetry (SWV) measurements were performed on a CHI 852C electrochemical workstation (CH Instruments, Shanghai, China), which is composed of a conventional three-electrode configuration containing the modified AuE working electrode, an Ag/AgCl (3 M KCl) reference electrode, and a platinum wire counter electrode. The SWV measurements were investigated by scanning the potential from −0.5 V to 0 V with a step potential of 4 mV, a frequency of 25 Hz and amplitude of 25 mV.
Results and discussion The amplified and signal-on electrochemical DNA sensing approach by target-induced reconfiguration of dsDNA probes is illustrated in Scheme 1. The dsDNA probes (SP–AP) are selfassembled on the AuE through the formation of Au–S bonds, followed by surface blocking with MCH to prepare the sensing surface. The dsDNA probes are formed by partial hybridization between SP and AP, leaving the 7- and 9-nt single-stranded
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Scheme 1 Schematic diagram of target recycling amplified and signalon electrochemical DNA detection.
region at one 3′-terminus and the other 3′-terminus, respectively. The dsDNA probes with both protruding 3′-termini are thus inactive substrates because Exo III catalyzes the stepwise removal of mononucleotides from blunt or recessed 3′-hydroxyl terminus of duplex DNA while it is inactive on ssDNA or protruding 3′ terminus of duplex DNA. The dsDNA probes immobilized on the electrode surface with 16 complementary bases thus force the MB tags away from the electrode surface, leading to a small background current without the presence of the target DNA. Conversely, when the target DNA is incubated with the sensor in the presence of Exo III, it hybridizes with the 9-nt single-stranded region of the dsDNA probes and reconfigures the dsDNA probes into duplexes with recessed 3′-termini, which are active substrates for Exo III. Subsequently, Exo III catalytically digests AP of the reconfigured duplexes, resulting in the release of the target DNA and the formation of single stranded SP. The single stranded SP further folds into hairpin structures in the presence of Mg2+ and brings the MB label into proximity with the electrode, which facilitates the electron transfer from MB to the electrode during potential scan and generates an enhanced current response for signal-on detection of the target DNA. Meanwhile, the released target DNA can again hybridize with the dsDNA probes and initiate another enzymatic digestion cycle, leading to the cyclic reuse of the target DNA and the formation of many hairpin structures that produce a significantly amplified current signal output for achieving sensitive detection of the target DNA. As can be seen from Fig. 1, the stepwise modification on the gold electrode is accompanied by a change in the current responses, as well as the separation between the cathodic and anodic peak of the redox probe [Fe(CN)6]3−/4−. For the bare AuE, there is a pair of well-defined redox peaks (curve a), showing the excellent electron-transfer kinetics of [Fe(CN)6]3−/4−. When dsDNA/MCH is immobilized on the electrode (note: to avoid current interference, the SP used here is free of the MB label), the current response decreases accompanied by an increase in the peak potential separation (ΔEp) between the cathodic and anodic peaks (curve b), which can be ascribed to the repellence of the negatively charged
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Fig. 1 Typical cyclic voltammograms recorded on different modified electrodes: (a) bare AuE, (b) MCH/dsDNA/AuE, (c) MCH/dsDNA/AuE after incubation with 0.5 nM target DNA, (d) MCH/dsDNA/AuE after incubation with 0.5 nM target and 16 U Exo III. CV measurements were performed in 1 mM [Fe(CN)6]3−/4− containing 0.1 M KCl solution by scanning the potential from −0.1 V to 0.6 V at a scan rate of 50 mV s−1.
redox species, [Fe(CN)6]3−/4−, from approaching the electrode surface by the negatively charged phosphate backbones of the duplex DNA probes. After further incubation with the target DNA in the absence of Exo III, the current response decreases and the ΔEp enlarges (curve c) because of the introduction of more negative charges from the hybridized target DNA. However, the current response is partially recovered (curve d) to where the current is at a higher intensity and a smaller ΔEp after treatment with the target DNA in the presence of Exo III, which is owing to Exo III-catalyzed digestion and the removal of AP from the electrode surface. The signal amplification capability of our protocol was confirmed by comparing the SWV responses of the sensor for the presence and absence of target DNA with and without Exo III. As shown in Fig. 2, the MCH/dsDNA/AuE electrode exhibits a small current response (curve a), which is due to the electron tunneling from the MB labels to the electrode. The incubation of the sensor with the target DNA (0.5 nM) causes insignificant change in the current response (curve b) because the distance between the MB labels and the electrode remains almost unchanged upon the hybridization of the target DNA with the
Fig. 2 Typical SWV responses of the sensing electrodes after incubation without (a) and with (b) the target DNA (0.5 nM) in the absence (c) and presence (d) of Exo III (16 U). SWV measurements were performed in 20 mM Tris buffer with a step potential of 4 mV, a frequency of 25 Hz and an amplitude of 25 mV by scanning the potential from −0.50 V to 0 V.
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dsDNA probes. Similarly, the addition of Exo III (16 U) to the sensor without the presence of the target DNA leads to negligible change in the current response (curve c) due to the fact that the dsDNA probes are inactive substrates for catalytic digestion by Exo III in the absence of the target DNA. Importantly, the incubation of the sensor with the target DNA (0.5 nM target) and Exo III (16 U) results in a significant increase in the current response (curve d), which is due to the cyclic cleavage of AP by Exo III upon the hybridization with the target DNA and the formation of plenty of hairpin structures on the sensor electrode as discussed previously. These experimental comparisons here clearly indicate the signal enhancement with the involvement of the Exo III in the assay method. To obtain optimal experimental conditions, we first optimized the immobilization concentration of dsDNA probes at 0.1 µM, 0.2 µM and 0.5 µM, respectively, for the detection of target DNA (0.5 nM). As shown in Fig. 3A, the background current increases accordingly with the increasing immobilization concentration of the dsDNA probes, owing to the increased amount of electroactive species of MB on the electrode surface. However, a low concentration of dsDNA probes at 0.1 µM results in the lowest SWV current response in the presence of the target DNA and the smallest signal-to-noise ratio (i/i0, i and i0 correspond to the SWV peak currents in the presence and absence of the target DNA, respectively) because of the decreased formation of hairpin structures. Furthermore, high immobilization concentration of the dsDNA probes (0.5 µM) shows a small increase in the current response than that of the one with the immobilization concentration of 0.2 µM in the presence of the target DNA, due to the steric hindrance for the formation of hairpin structures at high probe concentrations while the high background current obviously reduces the value of i/i0. According to the results, a medium density (0.2 μM) of dsDNA exhibits the best i/i0 value and is selected as the optimal immobilization concentration for subsequent
Fig. 3 Optimization of (A) the immobilization concentration of dsDNA (with the enzymatic reaction time for 160 min and the amount of Exo III at 16 U), (B) the enzymatic reaction time (with the immobilization concentration of dsDNA at 0.2 μM and the amount of Exo III at 16 U) and (C) the amount of Exo III (with the immobilization concentration of dsDNA at 0.2 μM and the enzymatic reaction time for 120 min) for amplified detection of target DNA (0.5 nM) by using the proposed sensing method. Error bars: SD, n = 3. Other conditions as in Fig. 2.
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experiments. Moreover, both the enzymatic reaction time and the amount of the Exo III can also potentially affect the assay performance for the detection of target DNA. From Fig. 3B, we can see that the current peak increases with the extension of the incubation time from 20 to 120 min and reaches a plateau at 120 min. Therefore, 120 min is selected as the optimal incubation time. In Fig. 3C, the current increases with the increasing amount of Exo III from 2 to 10 U and remains stable in the range from 10 to 20 U, thus the amount of 10 U Exo III is set as the optimal experiment conditions. In order to investigate the dependence of the peak current upon the concentration of the target DNA, the sensors were incubated with different concentrations of target DNA under the optimized conditions. From Fig. 4A, we can see that the blank test (in the absence of the target DNA) shows a small background signal (curve a) and the SWV peak current increases with elevated concentration of the target DNA from 0.5 pM to 50 nM. As shown in Fig. 4B, the current response is proportional to the logarithm of the target DNA concentration in the investigated range and the detection limit is estimated to be 0.24 pM according to the signal-to-noise ratio of three. The linear regression equation is determined to be i = 1.365 + 0.0942 log c with a correlation coefficient (R2) of 0.9952. Furthermore, six repetitive measurements of target DNA at 0.5 nM yielded a relative standard deviation of 7.8%, indicating good reproducibility of the sensor. The selectivity of the proposed biosensor was further investigated by using the target DNA (0.5 nM) against the control sequences, sDNA and nDNA (5 nM, at 10-fold excess than the target DNA). From the investigation (Fig. 5), we can see that the change in the current response in the presence of 5 nM nDNA (curve b) is negligible compared to the blank test (curve a, in the absence of the target DNA). Despite the apparent increase in the peak current for sDNA (curve c) compared with the blank experiment, the presence of a much lower concentration of the target DNA (0.5 nM, curve d) yields a significantly higher SWV peak current. The above results demonstrate that the developed detection method is highly selective toward target DNA against other control sequences. To evaluate the analytical reliability and real application of the proposed method, recovery experiments were carried out
Fig. 4 (A) Typical SWV responses of the proposed sensor for different concentrations of the target DNA: (a) 0, (b) 0.5 pM, (c) 5 pM, (d) 50 pM, (e) 0.5 nM, (f ) 5 nM, and (g) 50 nM. (B) The resulting calibration plot of i vs. log c. Error bars, SD, n = 3. Other conditions as in Fig. 2.
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Notes and references
Fig. 5 Specificity analysis of the assay protocol for the target DNA (0.5 nM, curve d) against other control sequences: blank (curve a), sDNA (5 nM, curve c) and nDNA (5 nM, curve b). Error bars, SD, n = 3. Other conditions as in Fig. 2.
Table 1 Determination of the target DNA in human serum with the proposed method (n = 6)
Sample
Added (nM)
Found (nM)
Rate of recovery (%)
RSD (%)
1 2 3
0.5 5.0 50.0
0.53 5.1 48.0
102–106 96–103 98–101
4.8 3.8 4.5
by analyzing the target DNA in human serum samples. Various concentrations of the target DNA were added to 10% human serum samples (diluted with buffer), followed by quantitating the target DNA with the developed sensor. As shown in Table 1, the recoveries fall in the range between 96% and 106%, indicating the potential of the proposed method for DNA detection in human serum samples. In conclusion, we have demonstrated a simple, sensitive and signal-on electrochemical method for the detection of DNA based on the target-induced reconfiguration of the dsDNA probes and enzyme-assisted target recycling amplification. With the combination of the signal-on sensing scheme and effective target recycling amplification, the detection of the sub-picomolar level of the hereditary tyrosinemia type I gene can be achieved. Besides, the developed method exhibits high selectivity and can be used to monitor the target gene in human serum samples. With the advantage of the sequence independence of the Exo III nuclease, the developed method thus holds great potential for convenient and sensitive detection of a wide range of nucleic acid biomarkers.
Acknowledgements This work is supported by the National Natural Science Foundation of China (no. 21275004, and 21275119), the New Century Excellent Talent Program of MOE (NCET-12-0932) and Fundamental Research Funds for the Central Universities (XDJK2014A012).
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Target-induced reconfiguration of DNA probes for recycling amplification and signal-on electrochemical detection of hereditary tyrosinemia type I gene Baoting Dou, Cuiyun Yang, Yaqin Chai, Ruo Yuan and Yun Xiang* By coupling target DNA-induced reconfiguration of the dsDNA probes with enzyme-assisted target recycling amplification, we describe the development of a signal-on electrochemical sensing approach for sensitive detection of hereditary tyrosinemia type I gene. The dsDNA probes are self-assembled on the sensing electrode, and the addition of the target DNA reconfigures and switches the dsDNA probes into active substrates for exonuclease III, which catalytically digests the probes and leads to cyclic reuse of the target DNA. The target DNA recycling and the removal of one of the ssDNA from the dsDNA probes by exonuclease III result in the formation of many hairpin structures on the sensor surface, which brings the electroactive methylene blue labels into proximity with the electrode and produces a significantly amplified
Received 19th May 2015, Accepted 26th June 2015
current response for sensitive detection of the target gene down to 0.24 pM. This method is also selective
DOI: 10.1039/c5an01006c
to discriminate single-base mismatch and can be employed to detect the target gene in human serum samples. With the demonstration for the detection of the target gene, we expect the developed method to
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be a universal sensitive sensing platform for the detection of different nucleic acid sequences.
Introduction The identification and detection of sequence specific DNA plays important roles in molecular diagnostics,1,2 forensic investigations,3 genetics therapy,4 and biomedical 5–8 development. Achieving high sensitivity and low detection limit is one of the major goals in developing different DNA sensing methods, and a variety of signal amplifications have been demonstrated to meet this goal. Traditional signal amplification methods including polymerase chain reaction (PCR)9 and rolling circle amplification (RCA)10,11 and so on can realize sensitive detection of DNA but are accompanied by the drawbacks of sophisticated instrumentation, easy contamination and high cost. This has led to the development of new signal amplification strategies for detecting low levels of DNA. The post-amplification strategies with the signals produced by hybridization events and the newly developed target cycling strategies have become the most common signal amplification platforms for the detection of DNA. The former relies on sequence specific hybridization recognition of the target DNA and the use of amplification labels such as enzymes,12,13 nano-
Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China. E-mail: [email protected]; Fax: +86-23-68252277; Tel: +86-23-68253172
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materials,14,15 DNA concatamers,16,17 and DNA biobarcodes18 to generate an amplified signal output. These amplification labels usually involve multiple assay steps and require the addition of many exogenous reagents. For example, nanomaterials provide the opportunity for amplifying hybridization signals of DNA biosensors by incorporating DNA with nanomaterials, due to their unique chemical and physical properties caused by their quantum confinement, surface, small size, and macro-quantum tunnel effects. However, these nanomaterials require complex fabrication processes and highly trained technicians.19–21 The target recycling amplification strategies, which involve the cyclic usage of the target molecules, have recently gained increased attention in DNA sensing. The achievement of target DNA recycling in these approaches is based on catalytic digestion of the hybridizedprobe sequences or displacement of the target DNA in the presence of a primer and polymerase upon the hybridizations between the target and probe sequences with the assistance of various nucleases. Without the requirement of a designated primer, the target recycling by nuclease catalytic digestion, including endonucleases (restriction enzymes)22–24 and exonucleases,25,26 can also achieve greatly enhanced sensitivity owing to the multiple hybridization events of the target sequences. Both endonucleases and exonucleases can digest the oligonucleotide probes hybridized with the target DNA selectively and release the target DNA to achieve target DNA
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recycling. Considering the fact that specific nucleotide sequences must be included in the oligonucleotide probes when using the endonucleases,22,27 the use of exonucleases for target DNA recycling without the requirement of specific sequences in the probes is more convenient, and a large number of target DNA recycling methods with the assistance of exonucleases have been developed to achieve sensitive detection of DNA in connection with fluorescent,28,29 electrochemical,30–32 surface plasmon resonance (SPR)33 or surface enhanced resonance Raman scattering (SERS)34,35 transduction means. Among these amplified detection schemes, the electrochemical approaches have received particular interest because of the intrinsic advantages of these techniques in terms of simplicity, low cost, portability and high sensitivity. Herein, based on a new target-induced reconfiguration strategy, we report on an enzyme-assisted target recycling amplification approach for sensitive electrochemical detection of hereditary tyrosinemia type I gene, a biomarker for the fumarylacetoacetate hydrolase gene mutation.36,37 The target DNAs hybridize with the dsDNA probes on the electrode surface and reconfigure the probes into active substrates for exonuclease, which cleaves one of the ssDNA strands in the DNA duplex probes and recycles the target DNA, generating amplified and “turn-on” electrochemical signals for detecting the hereditary tyrosinemia type I gene down to the sub-picomolar level.
Experimental Materials and reagents Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and 6-mercapto-1-hexanol (MCH) were obtained from SigmaAldrich (St. Louis, MO, USA). Exonuclease III (Exo III) and 10× NEBuffer 1 (1× NEBuffer 1, 10 mM bis-tris-propane-HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.0) were purchased from New England Biolabs (Beijing, China). The HPLC-purified oligonucleotides with sequences listed below were ordered from Sangon Biotech. Co., Ltd (Shanghai, China). Thiolated and methylene blue (MB)-modified hairpin DNA signal probe (SP): 5′-MB-(CH2)7-GAC GCT AAT AAT AGC ATA GCG TC-(CH2)6-SH-3′; assistant probe (AP): 5′-TGC TAT TAT TAG CGT CAT TCA CCG G-3′; target DNA (target): 5′-CCG GTG AAT ATC TGG-3′; single-base mismatched DNA (sDNA): 5′-CCG GC̲G AAT ATC TGG-3′; non-complementary DNA (nDNA): 5′TAT TCA GCG GCA CTA-3′. The bold and italic sequences, respectively, correspond to the complementary sequences between SP/AP and AP/target. All other chemicals used were of analytical reagent grade and all aqueous solutions were prepared with deionized water (specific resistance of 18 MΩ cm). Pretreatment of the gold electrode Prior to use, the 3.0 mm diameter gold electrodes (AuEs, CH Instruments Inc., Shanghai, China) were soaked in a freshly
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prepared Piranha solution (mixture of 98% H2SO4 and 30% H2O2 at a volume ratio of 3 : 1) for 0.5 hour. After rinsing thoroughly with water, the AuEs were polished with 0.3 and 0.05 µm aluminum slurry for 5 min separately, followed by sonicating in pure water, ethanol and pure water for 5 minutes, respectively, to remove the residual alumina powder. Next, each AuE was electrochemically cleaned in 0.5 M H2SO4 with the potential scanning from 0.3 to 1.5 V until stable characteristic voltammetric peaks were obtained. Following being dried with nitrogen, the cleaned electrodes were immediately used for probe immobilization. Sensor fabrication Firstly, the dsDNA probes (SP–AP) were obtained by annealing the mixture of SP (2.0 µM) and AP (2.2 µM) in 10 mM phosphate buffer solution (0.1 M NaCl, pH 7.4) at 85 °C for 10 minutes, followed by cooling down to 25 °C in 1 h. Subsequently, the annealed dsDNA probes (0.2 µM) were incubated with TCEP (10 mM) for 1 hour to reduce the disulfide bond and the MB moiety of SP. Next, droplets of 10 µL of the reduced probes were cast on the pretreated AuE surface and incubated overnight. After that, the electrodes were rinsed with water and then blocked with 1 mM MCH for 2 h to prepare the MCH/dsDNA/AuE modified electrodes. The modified electrodes were then immersed in 20 mM Tris buffer (140 mM NaCl, 5 mM MgCl2, pH 7.4) and allowed to equilibrate for 1 h, followed by measuring the responses of MB by square wave voltammetry (SWV) in 20 mM Tris buffer (140 mM NaCl, 5 mM MgCl2, pH 7.4) every fifteen minutes until the variation in MB signals was less than 1%. After getting the background current, the modified electrodes were incubated with 10 µL of solution containing various concentrations of target DNA and 10 U Exo III at 37 °C for 120 min. The sensors were further measured by SWV to obtain the signal responses. Electrochemical measurements Cyclic voltammetry (CV) and square wave voltammetry (SWV) measurements were performed on a CHI 852C electrochemical workstation (CH Instruments, Shanghai, China), which is composed of a conventional three-electrode configuration containing the modified AuE working electrode, an Ag/AgCl (3 M KCl) reference electrode, and a platinum wire counter electrode. The SWV measurements were investigated by scanning the potential from −0.5 V to 0 V with a step potential of 4 mV, a frequency of 25 Hz and amplitude of 25 mV.
Results and discussion The amplified and signal-on electrochemical DNA sensing approach by target-induced reconfiguration of dsDNA probes is illustrated in Scheme 1. The dsDNA probes (SP–AP) are selfassembled on the AuE through the formation of Au–S bonds, followed by surface blocking with MCH to prepare the sensing surface. The dsDNA probes are formed by partial hybridization between SP and AP, leaving the 7- and 9-nt single-stranded
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Scheme 1 Schematic diagram of target recycling amplified and signalon electrochemical DNA detection.
region at one 3′-terminus and the other 3′-terminus, respectively. The dsDNA probes with both protruding 3′-termini are thus inactive substrates because Exo III catalyzes the stepwise removal of mononucleotides from blunt or recessed 3′-hydroxyl terminus of duplex DNA while it is inactive on ssDNA or protruding 3′ terminus of duplex DNA. The dsDNA probes immobilized on the electrode surface with 16 complementary bases thus force the MB tags away from the electrode surface, leading to a small background current without the presence of the target DNA. Conversely, when the target DNA is incubated with the sensor in the presence of Exo III, it hybridizes with the 9-nt single-stranded region of the dsDNA probes and reconfigures the dsDNA probes into duplexes with recessed 3′-termini, which are active substrates for Exo III. Subsequently, Exo III catalytically digests AP of the reconfigured duplexes, resulting in the release of the target DNA and the formation of single stranded SP. The single stranded SP further folds into hairpin structures in the presence of Mg2+ and brings the MB label into proximity with the electrode, which facilitates the electron transfer from MB to the electrode during potential scan and generates an enhanced current response for signal-on detection of the target DNA. Meanwhile, the released target DNA can again hybridize with the dsDNA probes and initiate another enzymatic digestion cycle, leading to the cyclic reuse of the target DNA and the formation of many hairpin structures that produce a significantly amplified current signal output for achieving sensitive detection of the target DNA. As can be seen from Fig. 1, the stepwise modification on the gold electrode is accompanied by a change in the current responses, as well as the separation between the cathodic and anodic peak of the redox probe [Fe(CN)6]3−/4−. For the bare AuE, there is a pair of well-defined redox peaks (curve a), showing the excellent electron-transfer kinetics of [Fe(CN)6]3−/4−. When dsDNA/MCH is immobilized on the electrode (note: to avoid current interference, the SP used here is free of the MB label), the current response decreases accompanied by an increase in the peak potential separation (ΔEp) between the cathodic and anodic peaks (curve b), which can be ascribed to the repellence of the negatively charged
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Fig. 1 Typical cyclic voltammograms recorded on different modified electrodes: (a) bare AuE, (b) MCH/dsDNA/AuE, (c) MCH/dsDNA/AuE after incubation with 0.5 nM target DNA, (d) MCH/dsDNA/AuE after incubation with 0.5 nM target and 16 U Exo III. CV measurements were performed in 1 mM [Fe(CN)6]3−/4− containing 0.1 M KCl solution by scanning the potential from −0.1 V to 0.6 V at a scan rate of 50 mV s−1.
redox species, [Fe(CN)6]3−/4−, from approaching the electrode surface by the negatively charged phosphate backbones of the duplex DNA probes. After further incubation with the target DNA in the absence of Exo III, the current response decreases and the ΔEp enlarges (curve c) because of the introduction of more negative charges from the hybridized target DNA. However, the current response is partially recovered (curve d) to where the current is at a higher intensity and a smaller ΔEp after treatment with the target DNA in the presence of Exo III, which is owing to Exo III-catalyzed digestion and the removal of AP from the electrode surface. The signal amplification capability of our protocol was confirmed by comparing the SWV responses of the sensor for the presence and absence of target DNA with and without Exo III. As shown in Fig. 2, the MCH/dsDNA/AuE electrode exhibits a small current response (curve a), which is due to the electron tunneling from the MB labels to the electrode. The incubation of the sensor with the target DNA (0.5 nM) causes insignificant change in the current response (curve b) because the distance between the MB labels and the electrode remains almost unchanged upon the hybridization of the target DNA with the
Fig. 2 Typical SWV responses of the sensing electrodes after incubation without (a) and with (b) the target DNA (0.5 nM) in the absence (c) and presence (d) of Exo III (16 U). SWV measurements were performed in 20 mM Tris buffer with a step potential of 4 mV, a frequency of 25 Hz and an amplitude of 25 mV by scanning the potential from −0.50 V to 0 V.
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dsDNA probes. Similarly, the addition of Exo III (16 U) to the sensor without the presence of the target DNA leads to negligible change in the current response (curve c) due to the fact that the dsDNA probes are inactive substrates for catalytic digestion by Exo III in the absence of the target DNA. Importantly, the incubation of the sensor with the target DNA (0.5 nM target) and Exo III (16 U) results in a significant increase in the current response (curve d), which is due to the cyclic cleavage of AP by Exo III upon the hybridization with the target DNA and the formation of plenty of hairpin structures on the sensor electrode as discussed previously. These experimental comparisons here clearly indicate the signal enhancement with the involvement of the Exo III in the assay method. To obtain optimal experimental conditions, we first optimized the immobilization concentration of dsDNA probes at 0.1 µM, 0.2 µM and 0.5 µM, respectively, for the detection of target DNA (0.5 nM). As shown in Fig. 3A, the background current increases accordingly with the increasing immobilization concentration of the dsDNA probes, owing to the increased amount of electroactive species of MB on the electrode surface. However, a low concentration of dsDNA probes at 0.1 µM results in the lowest SWV current response in the presence of the target DNA and the smallest signal-to-noise ratio (i/i0, i and i0 correspond to the SWV peak currents in the presence and absence of the target DNA, respectively) because of the decreased formation of hairpin structures. Furthermore, high immobilization concentration of the dsDNA probes (0.5 µM) shows a small increase in the current response than that of the one with the immobilization concentration of 0.2 µM in the presence of the target DNA, due to the steric hindrance for the formation of hairpin structures at high probe concentrations while the high background current obviously reduces the value of i/i0. According to the results, a medium density (0.2 μM) of dsDNA exhibits the best i/i0 value and is selected as the optimal immobilization concentration for subsequent
Fig. 3 Optimization of (A) the immobilization concentration of dsDNA (with the enzymatic reaction time for 160 min and the amount of Exo III at 16 U), (B) the enzymatic reaction time (with the immobilization concentration of dsDNA at 0.2 μM and the amount of Exo III at 16 U) and (C) the amount of Exo III (with the immobilization concentration of dsDNA at 0.2 μM and the enzymatic reaction time for 120 min) for amplified detection of target DNA (0.5 nM) by using the proposed sensing method. Error bars: SD, n = 3. Other conditions as in Fig. 2.
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experiments. Moreover, both the enzymatic reaction time and the amount of the Exo III can also potentially affect the assay performance for the detection of target DNA. From Fig. 3B, we can see that the current peak increases with the extension of the incubation time from 20 to 120 min and reaches a plateau at 120 min. Therefore, 120 min is selected as the optimal incubation time. In Fig. 3C, the current increases with the increasing amount of Exo III from 2 to 10 U and remains stable in the range from 10 to 20 U, thus the amount of 10 U Exo III is set as the optimal experiment conditions. In order to investigate the dependence of the peak current upon the concentration of the target DNA, the sensors were incubated with different concentrations of target DNA under the optimized conditions. From Fig. 4A, we can see that the blank test (in the absence of the target DNA) shows a small background signal (curve a) and the SWV peak current increases with elevated concentration of the target DNA from 0.5 pM to 50 nM. As shown in Fig. 4B, the current response is proportional to the logarithm of the target DNA concentration in the investigated range and the detection limit is estimated to be 0.24 pM according to the signal-to-noise ratio of three. The linear regression equation is determined to be i = 1.365 + 0.0942 log c with a correlation coefficient (R2) of 0.9952. Furthermore, six repetitive measurements of target DNA at 0.5 nM yielded a relative standard deviation of 7.8%, indicating good reproducibility of the sensor. The selectivity of the proposed biosensor was further investigated by using the target DNA (0.5 nM) against the control sequences, sDNA and nDNA (5 nM, at 10-fold excess than the target DNA). From the investigation (Fig. 5), we can see that the change in the current response in the presence of 5 nM nDNA (curve b) is negligible compared to the blank test (curve a, in the absence of the target DNA). Despite the apparent increase in the peak current for sDNA (curve c) compared with the blank experiment, the presence of a much lower concentration of the target DNA (0.5 nM, curve d) yields a significantly higher SWV peak current. The above results demonstrate that the developed detection method is highly selective toward target DNA against other control sequences. To evaluate the analytical reliability and real application of the proposed method, recovery experiments were carried out
Fig. 4 (A) Typical SWV responses of the proposed sensor for different concentrations of the target DNA: (a) 0, (b) 0.5 pM, (c) 5 pM, (d) 50 pM, (e) 0.5 nM, (f ) 5 nM, and (g) 50 nM. (B) The resulting calibration plot of i vs. log c. Error bars, SD, n = 3. Other conditions as in Fig. 2.
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Notes and references
Fig. 5 Specificity analysis of the assay protocol for the target DNA (0.5 nM, curve d) against other control sequences: blank (curve a), sDNA (5 nM, curve c) and nDNA (5 nM, curve b). Error bars, SD, n = 3. Other conditions as in Fig. 2.
Table 1 Determination of the target DNA in human serum with the proposed method (n = 6)
Sample
Added (nM)
Found (nM)
Rate of recovery (%)
RSD (%)
1 2 3
0.5 5.0 50.0
0.53 5.1 48.0
102–106 96–103 98–101
4.8 3.8 4.5
by analyzing the target DNA in human serum samples. Various concentrations of the target DNA were added to 10% human serum samples (diluted with buffer), followed by quantitating the target DNA with the developed sensor. As shown in Table 1, the recoveries fall in the range between 96% and 106%, indicating the potential of the proposed method for DNA detection in human serum samples. In conclusion, we have demonstrated a simple, sensitive and signal-on electrochemical method for the detection of DNA based on the target-induced reconfiguration of the dsDNA probes and enzyme-assisted target recycling amplification. With the combination of the signal-on sensing scheme and effective target recycling amplification, the detection of the sub-picomolar level of the hereditary tyrosinemia type I gene can be achieved. Besides, the developed method exhibits high selectivity and can be used to monitor the target gene in human serum samples. With the advantage of the sequence independence of the Exo III nuclease, the developed method thus holds great potential for convenient and sensitive detection of a wide range of nucleic acid biomarkers.
Acknowledgements This work is supported by the National Natural Science Foundation of China (no. 21275004, and 21275119), the New Century Excellent Talent Program of MOE (NCET-12-0932) and Fundamental Research Funds for the Central Universities (XDJK2014A012).
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