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A novel electrochemical biosensor for HIV-related DNA detection based on toehold strand displacement reaction and cruciform DNA crystal
Journal of Electroanalytical Chemistry 822 (2018) 66–72
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Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem
A novel electrochemical biosensor for HIV-related DNA detection based on toehold strand displacement reaction and cruciform DNA crystal Yushu Hua, Hongwei Lib, Jianbo Lia, a b
T
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Department of Forensic Medicine, Faculty of Basic Medical Sciences, Chongqing Medical University, Chongqing 400016, China Physical Evidence Identification Center, Chongqing Municipal Public Security Bureau, Chongqing 400707, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Electrochemical biosensor HIV-related DNA Early diagnosis Toehold strand displacement reaction Cruciform DNA crystal Biosensing strategy
A sensitive and specific electrochemical biosensor was developed for quantitative detection of HIV-related DNA by integrating toehold strand displacement reaction (TSDR) with cruciform DNA crystal. The TSDR system can be specifically triggered by HIV-related DNA generating plentiful double-stranded DNA products and releasing the target DNA to undergo an additional cycle of amplification. The hybridization reaction of double-stranded DNA products with capture probes on Au electrode surface makes the binding sites of cruciform DNA crystal available. After the attachment of cruciform DNA crystal and the employment of streptavidinylated alkaline phosphatase, a significantly amplified electrochemical signal is obtained. This strategy was applied to the highly sensitive detection of HIV-related DNA in a linear range from 1 pM to 100 nM and a detection limit as low as 0.21 pM was achieved. The proposed strategy can be accomplished without the employment of any enzymes or intensive purification. In addition, this method has good selectivity and anti-interference ability, which could be applied in complex samples with comparable analytical performance. For these advantages, the proposed strategy holds great potential for rapid and early clinical diagnosis of HIV infection.
1. Introduction Human immunodeficiency virus (HIV) is a retrovirus that attacks the host's immune system making it unable to resist many diseases and resulting in death. Thus, early diagnosis and clinical therapy of HIV infection can greatly reduce mortality rates [1]. At present, serologic tests including enzyme-linked immunosorbent assay (ELISA) test and western blot (WB) assay are the common methods for clinical diagnosis of HIV infection [2,3]. However, serologic tests are unable to identify the early stage of HIV infection because of low antibody levels, which is known as the window period [4,5]. Unlike serology tests, genetictesting is independent of the presence of HIV antibodies and HIV virus can be detected once the patient is infected. Thus, the detection of highly sensitive and specific HIV-related gene can efficiently narrow the window period. The existing methods for DNA detection is diversified, such as PCR [6], southern blotting [7], microarray [8]. These methods are reliable but usually time-consuming, complicated, or require expensive instruments. At present, many biosensing strategies have been utilized to detect nucleic acid with high sensitivity, selectivity and simplicity, such as colorimetry [9,10], fluorescence [11,12], electrochemistry [13,14] and fluorescence resonance energy-transfer (FRET) [15,16]. Among
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Corresponding author. E-mail address: [email protected] (J. Li).
https://doi.org/10.1016/j.jelechem.2018.05.011 Received 2 April 2018; Received in revised form 5 May 2018; Accepted 11 May 2018 1572-6657/ © 2018 Elsevier B.V. All rights reserved.
them, electrochemical sensors have drawn intense attention due to the inherent advantages, including simple equipment, easy controllability, less sample consumption, and cost effective. To improve the sensitivity of electrochemical biosensor, various amplification techniques have been reported, involving bio-enzymes [17,18], metal nanoparticles [19,20], and DNA-fueled molecular machine [21,22]. In comparison with enzymes and metal nanoparticles based strategies, signal amplification strategies based on DNA nanotechnology are characteristic of simple materials, easy synthesis, good stability and excellent biocompatibility [23–25]. As a new class of target recycling strategy, toehold strand displacement reaction (TSDR) offers powerful design techniques for homogenous detection [26,27]. TSDR is a kind of metastable system consisting entirely of DNA oligonucleotides, which can be triggered by the addition of a specific singlestranded nucleic acid sequence [28]. TSDR is driven forward by entropy increase of the system in the absence of any enzymes, tags and stemloops which endows the system with easy operation and high sensitivity. The recognition of target sequence and nucleic acids complex through toehold region can successfully convert nucleic acid inputs into amplified outputs. So far, TSDR has been used as efficient signal amplification approach in nucleic acids detection [29,30], proteins analysis [31] and molecular imaging [32] fields. Furthermore, the
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detection system is a conventional three-electrode system composed of a platinum wire as auxiliary, an Ag/AgCl electrode as reference, and a 3-mm diameter Au electrode as working electrode. Gel electrophoresis was conducted on DYY-6C electrophoresis analyzer (Liuyi, Beijing, China) and visualized on Bio-Rad ChemDoc XRS (Bio-Rad, USA).
integration of TSDR with other systems can potentially improve the detection limits making TSDR more applicable to point-of-care or clinical diagnostics. With the development of DNA nanotechnology, DNA self-assembled nanomaterials have emerged as a favorable tool with the properties of both DNA and nanomaterials [33,34]. The self-assembly of DNA operates strictly according to Watson-Crick base pairing, affording the DNA nanomaterial high accuracy, good stability and programmability [35,36]. Owing to the intrinsic properties of DNA, DNA nanomaterial displays great diversity and versatility [37]. By assembling four DNA strands into a stable cruciform structure and incorporating different sticky-ends on the outside for hybridization, Seeman and his research group developed an artificial branched DNA crystal [38]. The formed four-way junction units can cohere based on the orientation of complementary sticky ends, producing larger frame crystals. Owing to the prominent flexibility, the constructed 2D/3D structures holds great promise for versatile applications in genomics, proteomics, diagnostics, and tissue engineering areas. In this work, we developed a sensitive electrochemical biosensor based on TSDR and cruciform DNA crystal for the detection of HIVrelated DNA. In TSDR system, the specific binding of target DNA through toehold region initiates the entire reaction, achieving onepot, catalytical signal amplification with no use of enzymes or precise thermal cycling. The TSDR double-strands products can directly hybridize with the detection probes on the Au electrode surface. After the successful attachment of TSDR double-strands products, the binding sites of cruciform DNA crystals are available. Then, the biotin labelled cruciform DNA crystals can favorably bind to electrode surface. By employing streptavidinylated alkaline phosphatase, the significantly amplified electrochemical signal can be easily obtained. Therefore, a simple and isothermal electrochemical biosensing strategy was achieved for highly specific and sensitive detection of HIV-related DNA, which is promising for rapid and early clinical diagnosis of HIV infection.
2.3. Preparation of probes The sequence of HIV-related DNA was designed according to the literatures [10,39]. DNA sequences of TSDR amplification system and cruciform DNA crystal were designed referring to recently published work [29,38]. The three-stranded complexes of TSDR were prepared with single strand Q, P and R at 1:1.2:1.2 M ratio in TSDR buffer. The solution was heated to 95 °C for 5 min and cooling to room temperature slowly. The cruciform DNA crystal was prepared through the successive assembly of four DNA strands. Single strands a, b, c and d were mixed in equimolar ratio in hybridization buffer with a final concentration of 1 μM. Then, the mixture was heated to 95 °C for 10 min and then chilled to 4 °C gradually. The probes were kept at 4 °C for further use. 2.4. Preparation of electrochemical biosensor The bare Au electrode was sequentially polished with 50 nm alumina slurries and ultrasonically treated in ultrapure water for 10 min. Then, the electrode was soaked in piranha solution for 10 min to eliminate other substances. After thoroughly rinsed with ultrapure water, the pretreated Au electrode was allowed to dry at room temperature. 10 μL of 0.2 μM thiolated capture probe was dropped on the surface of pretreated Au electrode and incubated overnight at 4 °C. After washed with washing buffer, the electrode was treated with 10 μL of 1 mM MCH for 1 h to occupy the left bare sites and obtain wellaligned DNA monolayer. The electrode was further rinsed with washing buffer and immersed into 2% BSA for 30 min to block the nonspecific binding sites on electrode surface to obtain the electrochemical DNA biosensor.
2. Experiment section 2.5. Electrochemical detection of HIV-related DNA 2.1. Materials and reagents The TSDR amplification system was carried out by mixing 2.5 μL complexes QPR (10 μM), 2.5 μL fuel strand F (10 μM) and varying concentrations of target in TSDR hybridization buffer to a final volume of 100 μL. Afterward, the mix was incubated at 37 °C for 30 min for further reaction. Then, 10 μL of resulted reaction mixture was dropped on the prepared electrode surface and incubated at 37 °C for 30 min. After rinsed with Tris-HCl buffer, 10 μL cruciform DNA crystal was added to the electrode surface and incubated at 37 °C for 30 min. Following rinsed by DEA buffer solution, the biosensor reacted with 10 μL of 1.25 μg mL−1 ST-AP at 37 °C for 30 min, and rinsed thoroughly with DEA buffer. The differential pulse voltammetry (DPV) measurement was performed in DEA buffer containing 1 mg mL−1 of α-NP substrate with modulation time of 0.05 s, interval time of 0.017 s, step potential of 5 mV, modulation amplitude of 70 mV and potential scan from 0.0 to +0.6 V.
6-mercaptohexanol (MCH), streptavidin-alkaline phosphatase (STALP), bovine serum albumin (BSA) and α-naphthylphosphate (α-NP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 20 bp DNA Ladder and agarose were purchased from Takara (Dalian, China). GoldView was purchased from SBS Genetech (Beijing, China). All other reagents were of analytical grade, and Millipore-Q water (≥18 MΩ cm) was used in all experiments. TSDR buffer (pH 7.0) contained 480 mM NaCl,10 mM Tris, and 5 mM MgCl2. Cruciform DNA crystal assembly buffer (pH 8.0) contained 30 mM sodium phosphate, 3 mM EDTA, 450 mM NaCl and 0.25% Triton 100. Tris–HCl buffer as washing buffer (pH 7.4) comprised 20 mM Tris, 5.0 mM MgCl2, 0.10 M NaCl and 0.05% Tween-20. Diethanolamine (DEA) buffer (pH 9.6) comprised 100 mM KCl, 0.1 M DEA and 1 M MgCl2. Piranha solution comprised 98% H2SO4 and 30% H2O2 (3:1 by volume). Caution: Piranha solutions are extremely aggressive. The use of safety goggles and a hood is strongly recommended. All DNA oligonucleotides were synthesized and purified by Sangon Biotechnology Co. Ltd. (Shanghai, China). The detailed sequences are listed in Table S1 (Supporting information). All oligonucleotides were dissolved in tris-ethylenediaminetetraacetic acid (TE) buffer (pH 8.0, 10 mM Tris-HCl, 1 mM ethylene diamine tetraacetic acid) and stored at −20 °C for future use.
2.6. Native polyacrylamide gel electrophoresis (PAGE) A 8% PAGE analysis of TSDR system and cruciform DNA crystal was carried out in 1 × TBE buffer (89 mM Tris-boric acid, 2 mM EDTA, pH 8.3) at 120 V constant voltage for 30 min. The gels were imaged using gel image system (Bio-Rad Laboratories, USA). 3. Results and discussion
2.2. Apparatus 3.1. Principle of the biosensing strategy All electrochemical measurements were performed on a CHI660D electrochemical workstation (Shanghai Chenhua Co., China). The
The principle for electrochemical detection of HIV-related DNA 67
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Scheme 1. (A) Scheme of the toehold strand displacement reaction, (B) Schematic illustration of the preparation of cruciform DNA crystal, (C) Schematic representation of HIV-related DNA electrochemical detection based on TSDR and cruciform DNA crystal.
phosphate backbone of the capture probe prevented the negative charged redox probe [Fe(CN)6]3−/4− from reaching the Au electrode and inhibited the transfer of interfacial charge. When the Au electrode surface was blocked with MCH and BSA, the Ret substantially increased (curve c), which was owing to the biomacromolecules retard electron transfer. At the presence of HIV-related DNA, TSDR was triggered, producing multiple double-stranded F-Q molecules, and resulting in Ret increase significantly (curve d). Afterwards, upon the cruciform DNA crystal binding onto the electrochemical sensor surface, the Ret further increased (curve e). These results were fairly consistent with those obtained from SWV measurements (Fig. 1B), in which the peak currents changed upon the assembly processes. Both results of EIS and SWV confirmed that the biosensor worked indeed as described in the principle scheme. In order to verify the designed TSDR strategy and the self-assembly of cruciform DNA crystal, the sequences and reaction products were characterized by 8% native polyacrylamide gel electrophoresis (PAGE). As depicted in Fig. 2A, the base number of HIV-related DNA (lane 2), fuel strand F (lane3) and three-stranded complex QPR (lane 4) derived from electrophoresis were consistent with our design. In the absence of target DNA, fuel strand F did not hybridize with the three-stranded complex QPR in lane 5. However, with the target DNA, complex QPR with high molecular weight were almost invisible and new band with lower mobility appeared (lane 6).The results in Fig. 2A indicated that the designed TSDR strategy could be successfully conducted. As shown in Fig. 2B, the bands in lane 2, lane 3, lane 4 and lane 5 represented strand a, strand b, strand c and strand d of cruciform DNA crystal, respectively. The bands in lane 6 were the annealing products of strand a and strand b. And the bands in lane 7 were annealing products of strand a, strand b and strand c. The bands in lane 6 and 7 indicated that two strands or three strands DNA cannot assemble to form cruciform DNA crystal. When the mixture of four strands was analyzed, the band with the slowest migration was observed (Lane 8), confirming the successful formation of cruciform DNA crystal. These results indicated the
based on TSDR amplification and cruciform DNA crystal is illustrated in Scheme 1. First, the thiolated capture probes are immobilized on the Au electrode surface though Au-thiol interaction. In the presence of HIVrelated DNA, strand P is released from the three-stranded complexes QPR through the toehold recognition. The release of strand P causes the exposure of new toehold in the middle of strand Q. Then, fuel strand F binds to the new toehold on strand Q and triggering a strand-displacement reaction among strand T, strand R and strand F. As a result, strand R is released and strand T is regenerated to trigger a new cycle. Up to now, in the presence of target DNA, the beacon complex QPR is converted to a double-stranded F-Q with the help of fuel strand F, releasing by-product strand R and P. Consequently, the formed doublestranded complex F-Q can specifically hybridize with capture probes on the Au electrode. After the hybridization, the complex F-Q acts as a linker for capturing cruciform DNA crystal. Then, the prepared cruciform DNA crystal can favorably bind to sensor surface, carrying numerous terminal biotin molecules to anchor the ST-ALP. Finally, ALP catalyzes the irreversible conversion of substrate α-NP to produce an amplified electrochemical signal, which is quantified by differential pulse voltammetry (DPV) for quantitative detection of HIV-related DNA. 3.2. Characterization of the biosensing strategy As shown in Fig. 1, electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV) measurements were adopted to characterize the established electrochemical biosensor. The EIS curves were obtained in 0.4 M KCl containing 0.5 mM Fe(CN)63−/4− (pH 6.1) and the semicircle diameter was equal to electron-transfer resistance (Ret) (Fig. 1A). In 0.4 M KCl containing 0.5 mM Fe(CN)63−/4−, bare Au electrode exhibited an almost straight line (curve a), reflecting the superior electrochemical conductivity. When the thiolated capture probe was assembled onto the bare Au electrode via Au-thiol binding, the Ret increased (curve b). The reason was that the negatively charged 68
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Fig. 1. EIS (A) and SWV (B) in 0.4 M KCl containing 0.5 mM Fe(CN)63−/4− at bare electrode (a), capture DNA modified electrode(b), MCH and BSA immobilized on the electrode surface(c), capture DNA modified electrode after hybridized with TSDR products (d) and with cruciform DNA crystal (e), respectively.
steady status at 250 nM, indicating that 250 nM was the optimal concentration of complex QPR. In addition, when fixed the concentration of cruciform DNA crystal, with the increasing concentration of DNA crystal, the signal-to-noise ratio also increased and tended to decrease at 1 μM, indicating that the higher concentration of DNA crystal could increase the nonspecific adsorption (Fig. 3D). Therefore, the concentration of cruciform DNA crystal was fixed at 1 μM for the following experiments. The error bars in the diagram stand for the standard deviations in three different results for the parallel tests.
feasibility of the designed TSDR strategy and cruciform DNA crystal nanoassembly directly. 3.3. Optimization of experimental conditions In order to obtain optimum analytical performance, crucial experimental variables were optimized, including the TSDR reaction time, reaction temperature, sequences concentration and DNA crystal concentration. The signal-to-noise ratio was utilized to assess the performance of the electrochemical biosensor. The TSDR process could be influenced obviously by the incubating time and temperature. The result of optimization of TSDR incubating time was depicted in Fig. 3A. The signal-to-noise ratio increased with the extension of incubating time. While the incubating time extended to 30 min, the signal generated by target DNA reached the largest and kept steady status. Hence, 30 min was chosen as the optimal reaction time. The reaction temperature was also examined from 4 to 48 °C (Fig. 3B), and the maximum signal-to-noise ratio were achieved at 37 °C. At low temperature, target DNA and complex QPR could not get a sufficient collision probability that significantly influenced the formation of double-stranded complex F-Q; at high temperature, the signal caused by non-stable complex QPR increased with the increase of incubating temperature. Thus, 37 °C was chosen as the optimal reaction temperature. In order to enhance detection sensitivity, the concentration of threestranded complex QPR and cruciform DNA crystal were also optimized. The effect of three-stranded complex QPR concentration on the signalto-noise ratio was investigated in Fig. 3C. With the augment of complex QPR concentration, the signal-to-noise ratio increased and then kept
3.4. Analytical performance of designed biosensor To evaluate the analytical performance of the established electrochemical biosensor, DPV responses to different concentrations of HIVrelated DNA were obtained under optimal conditions. As shown in Fig. 4A, The DPV peak current increased with the increase of target DNA concentration. The calibration plots showed a strong linear relationship between the peak currents and the logarithm of target DNA concentrations in the range from 1 pM to 100 nM (Fig. 4B). The resulting linear equation was ip (A) = 2.49 E−6 log CDNA (nM) + 1.05 E−5 with a correlation coefficient of 0.9978. According to the 3σ rule, the limit of detection (LOD) for target DNA was estimated to be 0.21 pM. For comparison, the analytical property of this method was compared with previously reported assays for nucleic acid detection. As shown in Table S2, the developed strategy had superior or comparable sensitivity to other nucleic acid detection strategies. The high sensitivity was attributed to the high efficiency of TSDR system and excellent signal amplification performance of cruciform DNA crystal. Hence, this
Fig. 2. (A) Native polyacrylamide gel electrophoresis results of the products and sequences of TSDR. Lane 1: 20 bp DNA ladder marker; Lane 2: HIV-related DNA; Lane 3: fuel strand F; Lane 4: three-stranded complex QPR; Lane 5: TSDR system without target DNA; Lane 6: TSDR system with target DNA. (B) Native polyacrylamide gel electrophoresis results of the products and sequences of cruciform DNA crystal. Lane 1: 20 bp DNA ladder marker; Lanes 2, 3, 4, 5: single strand a, b, c, d, respectively; Lane 6: annealing products of strand a and strand b; Lane 7: annealing products of strand a, strand b and strand c; Lane 8: cruciform DNA crystal.
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Fig. 3. Optimizations of experimental parameters: (A) evaluation of the effect of incubating time of TSDR, (B) evaluation of the effect of incubating temperature of TSDR, (C) evaluation of the effect of concentration of complex QPR, (D) evaluation of the effect of concentration of cruciform DNA crystal. The error bars represent the standard deviations calculated from three different spots.
biosensor can be applied to quantification of HIV-related DNA with a wide linear range and low detection concentration.
to that of the blank. However, in the presence of target DNA, there was a remarkable increase in the DPV current, which was about 5.5 times as that of SM and 11 times as that of DM and NC (Fig. 5B). These results indicated that the designed biosensor had remarkable specificity for the determination of HIV-related DNA. To estimate the repeatability of the developed biosensing strategy, the intra-assay precision of five different detections at one assay and inter-assay precision at five different assays for HIV-related DNA detection were measured, respectively. Consequently, the intra-assay coefficient of variation (CV) was 3.25% and the inter-assay CV was 4.43%, which manifested that this biosensor had an
3.5. Specificity and reproducibility of designed biosensor To evaluate the selectivity of the developed electrochemical method, four distinct control DNA sequences, including target DNA, single-base mismatch strand (SM), double-base mismatch strand (DM), and non-complementary sequence (NC) were used. As showed in Fig. 5A, DPV currents for 100 pM of DM and 100 pM of NC were similar
Fig. 4. (A) Typical DPV curves responding to 0, 0.001, 0.01, 0.1, 1, 10, and 100 nM of target DNA (from a to g). (B) Plot of DPV peak current vs. logarithm of target DNA concentration. 70
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Fig. 5. (A) Typical DPV curves and (B) DPV peak currents respectively respond to 100 pM of HIV-related DNA (a), single-base mismatched oligonucleotides (b), twobase mismatched oligonucleotides (c), non-complementary oligonucleotides (d), and blank (e).
acceptable reproducibility.
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3.6. Interfering effects of complex matrix To evaluate the interfering effects of complicated matrix on the proposed biosensor, the recovery assays were performed for the detection of target DNA in 10-fold diluted human serum samples. With a standard addition method, different amounts of target DNA were spiked into human serum samples for the assay. The recoveries were 94–106% from 10 pM to 10 nM (Table S3), indicating acceptable analytic performance in complex matrix. These good results were attributed to the diluted serum samples and several washing and separation steps to remove the nonspecific interferents. It implied that this method can be used for quantification of HIV-related DNA in complex biological matrix. 4. Conclusion To conclude, a simple and enzyme-free electrochemical biosensing strategy has been developed for HIV-related DNA detection by TSDR and cruciform DNA crystal cascade amplification. The enzyme-free strategy overcomes the disadvantages of traditional enzyme-based amplification and obtained satisfactory results. This electrochemical biosensor shows high sensitivity for the detection of HIV-related DNA with the dynamic linear range from 1 pM to 100 nM and the detection limit down to 0.21 pM. In addition, the designed biosensor possesses the advantages of simple material, excellent specificity, good reproducibility, and has been applied to analyze target DNA in human serum samples. Thus, this electrochemical biosensor would provide a promising platform for the detection of HIV-related DNA in biomedical research and early clinical diagnosis of HIV infection. Acknowledgements This work was funded by the Foundation for Science and Technology Research Project of Chongqing (KJ1502703). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jelechem.2018.05.011. References [1] E. Girardi, C.A. Sabin, A.D. Monforte, Late diagnosis of HIV infection: epidemiological features, consequences and strategies to encourage earlier testing, J. Acquir.
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Contents lists available at ScienceDirect
Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem
A novel electrochemical biosensor for HIV-related DNA detection based on toehold strand displacement reaction and cruciform DNA crystal Yushu Hua, Hongwei Lib, Jianbo Lia, a b
T
⁎
Department of Forensic Medicine, Faculty of Basic Medical Sciences, Chongqing Medical University, Chongqing 400016, China Physical Evidence Identification Center, Chongqing Municipal Public Security Bureau, Chongqing 400707, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Electrochemical biosensor HIV-related DNA Early diagnosis Toehold strand displacement reaction Cruciform DNA crystal Biosensing strategy
A sensitive and specific electrochemical biosensor was developed for quantitative detection of HIV-related DNA by integrating toehold strand displacement reaction (TSDR) with cruciform DNA crystal. The TSDR system can be specifically triggered by HIV-related DNA generating plentiful double-stranded DNA products and releasing the target DNA to undergo an additional cycle of amplification. The hybridization reaction of double-stranded DNA products with capture probes on Au electrode surface makes the binding sites of cruciform DNA crystal available. After the attachment of cruciform DNA crystal and the employment of streptavidinylated alkaline phosphatase, a significantly amplified electrochemical signal is obtained. This strategy was applied to the highly sensitive detection of HIV-related DNA in a linear range from 1 pM to 100 nM and a detection limit as low as 0.21 pM was achieved. The proposed strategy can be accomplished without the employment of any enzymes or intensive purification. In addition, this method has good selectivity and anti-interference ability, which could be applied in complex samples with comparable analytical performance. For these advantages, the proposed strategy holds great potential for rapid and early clinical diagnosis of HIV infection.
1. Introduction Human immunodeficiency virus (HIV) is a retrovirus that attacks the host's immune system making it unable to resist many diseases and resulting in death. Thus, early diagnosis and clinical therapy of HIV infection can greatly reduce mortality rates [1]. At present, serologic tests including enzyme-linked immunosorbent assay (ELISA) test and western blot (WB) assay are the common methods for clinical diagnosis of HIV infection [2,3]. However, serologic tests are unable to identify the early stage of HIV infection because of low antibody levels, which is known as the window period [4,5]. Unlike serology tests, genetictesting is independent of the presence of HIV antibodies and HIV virus can be detected once the patient is infected. Thus, the detection of highly sensitive and specific HIV-related gene can efficiently narrow the window period. The existing methods for DNA detection is diversified, such as PCR [6], southern blotting [7], microarray [8]. These methods are reliable but usually time-consuming, complicated, or require expensive instruments. At present, many biosensing strategies have been utilized to detect nucleic acid with high sensitivity, selectivity and simplicity, such as colorimetry [9,10], fluorescence [11,12], electrochemistry [13,14] and fluorescence resonance energy-transfer (FRET) [15,16]. Among
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Corresponding author. E-mail address: [email protected] (J. Li).
https://doi.org/10.1016/j.jelechem.2018.05.011 Received 2 April 2018; Received in revised form 5 May 2018; Accepted 11 May 2018 1572-6657/ © 2018 Elsevier B.V. All rights reserved.
them, electrochemical sensors have drawn intense attention due to the inherent advantages, including simple equipment, easy controllability, less sample consumption, and cost effective. To improve the sensitivity of electrochemical biosensor, various amplification techniques have been reported, involving bio-enzymes [17,18], metal nanoparticles [19,20], and DNA-fueled molecular machine [21,22]. In comparison with enzymes and metal nanoparticles based strategies, signal amplification strategies based on DNA nanotechnology are characteristic of simple materials, easy synthesis, good stability and excellent biocompatibility [23–25]. As a new class of target recycling strategy, toehold strand displacement reaction (TSDR) offers powerful design techniques for homogenous detection [26,27]. TSDR is a kind of metastable system consisting entirely of DNA oligonucleotides, which can be triggered by the addition of a specific singlestranded nucleic acid sequence [28]. TSDR is driven forward by entropy increase of the system in the absence of any enzymes, tags and stemloops which endows the system with easy operation and high sensitivity. The recognition of target sequence and nucleic acids complex through toehold region can successfully convert nucleic acid inputs into amplified outputs. So far, TSDR has been used as efficient signal amplification approach in nucleic acids detection [29,30], proteins analysis [31] and molecular imaging [32] fields. Furthermore, the
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detection system is a conventional three-electrode system composed of a platinum wire as auxiliary, an Ag/AgCl electrode as reference, and a 3-mm diameter Au electrode as working electrode. Gel electrophoresis was conducted on DYY-6C electrophoresis analyzer (Liuyi, Beijing, China) and visualized on Bio-Rad ChemDoc XRS (Bio-Rad, USA).
integration of TSDR with other systems can potentially improve the detection limits making TSDR more applicable to point-of-care or clinical diagnostics. With the development of DNA nanotechnology, DNA self-assembled nanomaterials have emerged as a favorable tool with the properties of both DNA and nanomaterials [33,34]. The self-assembly of DNA operates strictly according to Watson-Crick base pairing, affording the DNA nanomaterial high accuracy, good stability and programmability [35,36]. Owing to the intrinsic properties of DNA, DNA nanomaterial displays great diversity and versatility [37]. By assembling four DNA strands into a stable cruciform structure and incorporating different sticky-ends on the outside for hybridization, Seeman and his research group developed an artificial branched DNA crystal [38]. The formed four-way junction units can cohere based on the orientation of complementary sticky ends, producing larger frame crystals. Owing to the prominent flexibility, the constructed 2D/3D structures holds great promise for versatile applications in genomics, proteomics, diagnostics, and tissue engineering areas. In this work, we developed a sensitive electrochemical biosensor based on TSDR and cruciform DNA crystal for the detection of HIVrelated DNA. In TSDR system, the specific binding of target DNA through toehold region initiates the entire reaction, achieving onepot, catalytical signal amplification with no use of enzymes or precise thermal cycling. The TSDR double-strands products can directly hybridize with the detection probes on the Au electrode surface. After the successful attachment of TSDR double-strands products, the binding sites of cruciform DNA crystals are available. Then, the biotin labelled cruciform DNA crystals can favorably bind to electrode surface. By employing streptavidinylated alkaline phosphatase, the significantly amplified electrochemical signal can be easily obtained. Therefore, a simple and isothermal electrochemical biosensing strategy was achieved for highly specific and sensitive detection of HIV-related DNA, which is promising for rapid and early clinical diagnosis of HIV infection.
2.3. Preparation of probes The sequence of HIV-related DNA was designed according to the literatures [10,39]. DNA sequences of TSDR amplification system and cruciform DNA crystal were designed referring to recently published work [29,38]. The three-stranded complexes of TSDR were prepared with single strand Q, P and R at 1:1.2:1.2 M ratio in TSDR buffer. The solution was heated to 95 °C for 5 min and cooling to room temperature slowly. The cruciform DNA crystal was prepared through the successive assembly of four DNA strands. Single strands a, b, c and d were mixed in equimolar ratio in hybridization buffer with a final concentration of 1 μM. Then, the mixture was heated to 95 °C for 10 min and then chilled to 4 °C gradually. The probes were kept at 4 °C for further use. 2.4. Preparation of electrochemical biosensor The bare Au electrode was sequentially polished with 50 nm alumina slurries and ultrasonically treated in ultrapure water for 10 min. Then, the electrode was soaked in piranha solution for 10 min to eliminate other substances. After thoroughly rinsed with ultrapure water, the pretreated Au electrode was allowed to dry at room temperature. 10 μL of 0.2 μM thiolated capture probe was dropped on the surface of pretreated Au electrode and incubated overnight at 4 °C. After washed with washing buffer, the electrode was treated with 10 μL of 1 mM MCH for 1 h to occupy the left bare sites and obtain wellaligned DNA monolayer. The electrode was further rinsed with washing buffer and immersed into 2% BSA for 30 min to block the nonspecific binding sites on electrode surface to obtain the electrochemical DNA biosensor.
2. Experiment section 2.5. Electrochemical detection of HIV-related DNA 2.1. Materials and reagents The TSDR amplification system was carried out by mixing 2.5 μL complexes QPR (10 μM), 2.5 μL fuel strand F (10 μM) and varying concentrations of target in TSDR hybridization buffer to a final volume of 100 μL. Afterward, the mix was incubated at 37 °C for 30 min for further reaction. Then, 10 μL of resulted reaction mixture was dropped on the prepared electrode surface and incubated at 37 °C for 30 min. After rinsed with Tris-HCl buffer, 10 μL cruciform DNA crystal was added to the electrode surface and incubated at 37 °C for 30 min. Following rinsed by DEA buffer solution, the biosensor reacted with 10 μL of 1.25 μg mL−1 ST-AP at 37 °C for 30 min, and rinsed thoroughly with DEA buffer. The differential pulse voltammetry (DPV) measurement was performed in DEA buffer containing 1 mg mL−1 of α-NP substrate with modulation time of 0.05 s, interval time of 0.017 s, step potential of 5 mV, modulation amplitude of 70 mV and potential scan from 0.0 to +0.6 V.
6-mercaptohexanol (MCH), streptavidin-alkaline phosphatase (STALP), bovine serum albumin (BSA) and α-naphthylphosphate (α-NP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 20 bp DNA Ladder and agarose were purchased from Takara (Dalian, China). GoldView was purchased from SBS Genetech (Beijing, China). All other reagents were of analytical grade, and Millipore-Q water (≥18 MΩ cm) was used in all experiments. TSDR buffer (pH 7.0) contained 480 mM NaCl,10 mM Tris, and 5 mM MgCl2. Cruciform DNA crystal assembly buffer (pH 8.0) contained 30 mM sodium phosphate, 3 mM EDTA, 450 mM NaCl and 0.25% Triton 100. Tris–HCl buffer as washing buffer (pH 7.4) comprised 20 mM Tris, 5.0 mM MgCl2, 0.10 M NaCl and 0.05% Tween-20. Diethanolamine (DEA) buffer (pH 9.6) comprised 100 mM KCl, 0.1 M DEA and 1 M MgCl2. Piranha solution comprised 98% H2SO4 and 30% H2O2 (3:1 by volume). Caution: Piranha solutions are extremely aggressive. The use of safety goggles and a hood is strongly recommended. All DNA oligonucleotides were synthesized and purified by Sangon Biotechnology Co. Ltd. (Shanghai, China). The detailed sequences are listed in Table S1 (Supporting information). All oligonucleotides were dissolved in tris-ethylenediaminetetraacetic acid (TE) buffer (pH 8.0, 10 mM Tris-HCl, 1 mM ethylene diamine tetraacetic acid) and stored at −20 °C for future use.
2.6. Native polyacrylamide gel electrophoresis (PAGE) A 8% PAGE analysis of TSDR system and cruciform DNA crystal was carried out in 1 × TBE buffer (89 mM Tris-boric acid, 2 mM EDTA, pH 8.3) at 120 V constant voltage for 30 min. The gels were imaged using gel image system (Bio-Rad Laboratories, USA). 3. Results and discussion
2.2. Apparatus 3.1. Principle of the biosensing strategy All electrochemical measurements were performed on a CHI660D electrochemical workstation (Shanghai Chenhua Co., China). The
The principle for electrochemical detection of HIV-related DNA 67
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Scheme 1. (A) Scheme of the toehold strand displacement reaction, (B) Schematic illustration of the preparation of cruciform DNA crystal, (C) Schematic representation of HIV-related DNA electrochemical detection based on TSDR and cruciform DNA crystal.
phosphate backbone of the capture probe prevented the negative charged redox probe [Fe(CN)6]3−/4− from reaching the Au electrode and inhibited the transfer of interfacial charge. When the Au electrode surface was blocked with MCH and BSA, the Ret substantially increased (curve c), which was owing to the biomacromolecules retard electron transfer. At the presence of HIV-related DNA, TSDR was triggered, producing multiple double-stranded F-Q molecules, and resulting in Ret increase significantly (curve d). Afterwards, upon the cruciform DNA crystal binding onto the electrochemical sensor surface, the Ret further increased (curve e). These results were fairly consistent with those obtained from SWV measurements (Fig. 1B), in which the peak currents changed upon the assembly processes. Both results of EIS and SWV confirmed that the biosensor worked indeed as described in the principle scheme. In order to verify the designed TSDR strategy and the self-assembly of cruciform DNA crystal, the sequences and reaction products were characterized by 8% native polyacrylamide gel electrophoresis (PAGE). As depicted in Fig. 2A, the base number of HIV-related DNA (lane 2), fuel strand F (lane3) and three-stranded complex QPR (lane 4) derived from electrophoresis were consistent with our design. In the absence of target DNA, fuel strand F did not hybridize with the three-stranded complex QPR in lane 5. However, with the target DNA, complex QPR with high molecular weight were almost invisible and new band with lower mobility appeared (lane 6).The results in Fig. 2A indicated that the designed TSDR strategy could be successfully conducted. As shown in Fig. 2B, the bands in lane 2, lane 3, lane 4 and lane 5 represented strand a, strand b, strand c and strand d of cruciform DNA crystal, respectively. The bands in lane 6 were the annealing products of strand a and strand b. And the bands in lane 7 were annealing products of strand a, strand b and strand c. The bands in lane 6 and 7 indicated that two strands or three strands DNA cannot assemble to form cruciform DNA crystal. When the mixture of four strands was analyzed, the band with the slowest migration was observed (Lane 8), confirming the successful formation of cruciform DNA crystal. These results indicated the
based on TSDR amplification and cruciform DNA crystal is illustrated in Scheme 1. First, the thiolated capture probes are immobilized on the Au electrode surface though Au-thiol interaction. In the presence of HIVrelated DNA, strand P is released from the three-stranded complexes QPR through the toehold recognition. The release of strand P causes the exposure of new toehold in the middle of strand Q. Then, fuel strand F binds to the new toehold on strand Q and triggering a strand-displacement reaction among strand T, strand R and strand F. As a result, strand R is released and strand T is regenerated to trigger a new cycle. Up to now, in the presence of target DNA, the beacon complex QPR is converted to a double-stranded F-Q with the help of fuel strand F, releasing by-product strand R and P. Consequently, the formed doublestranded complex F-Q can specifically hybridize with capture probes on the Au electrode. After the hybridization, the complex F-Q acts as a linker for capturing cruciform DNA crystal. Then, the prepared cruciform DNA crystal can favorably bind to sensor surface, carrying numerous terminal biotin molecules to anchor the ST-ALP. Finally, ALP catalyzes the irreversible conversion of substrate α-NP to produce an amplified electrochemical signal, which is quantified by differential pulse voltammetry (DPV) for quantitative detection of HIV-related DNA. 3.2. Characterization of the biosensing strategy As shown in Fig. 1, electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV) measurements were adopted to characterize the established electrochemical biosensor. The EIS curves were obtained in 0.4 M KCl containing 0.5 mM Fe(CN)63−/4− (pH 6.1) and the semicircle diameter was equal to electron-transfer resistance (Ret) (Fig. 1A). In 0.4 M KCl containing 0.5 mM Fe(CN)63−/4−, bare Au electrode exhibited an almost straight line (curve a), reflecting the superior electrochemical conductivity. When the thiolated capture probe was assembled onto the bare Au electrode via Au-thiol binding, the Ret increased (curve b). The reason was that the negatively charged 68
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Fig. 1. EIS (A) and SWV (B) in 0.4 M KCl containing 0.5 mM Fe(CN)63−/4− at bare electrode (a), capture DNA modified electrode(b), MCH and BSA immobilized on the electrode surface(c), capture DNA modified electrode after hybridized with TSDR products (d) and with cruciform DNA crystal (e), respectively.
steady status at 250 nM, indicating that 250 nM was the optimal concentration of complex QPR. In addition, when fixed the concentration of cruciform DNA crystal, with the increasing concentration of DNA crystal, the signal-to-noise ratio also increased and tended to decrease at 1 μM, indicating that the higher concentration of DNA crystal could increase the nonspecific adsorption (Fig. 3D). Therefore, the concentration of cruciform DNA crystal was fixed at 1 μM for the following experiments. The error bars in the diagram stand for the standard deviations in three different results for the parallel tests.
feasibility of the designed TSDR strategy and cruciform DNA crystal nanoassembly directly. 3.3. Optimization of experimental conditions In order to obtain optimum analytical performance, crucial experimental variables were optimized, including the TSDR reaction time, reaction temperature, sequences concentration and DNA crystal concentration. The signal-to-noise ratio was utilized to assess the performance of the electrochemical biosensor. The TSDR process could be influenced obviously by the incubating time and temperature. The result of optimization of TSDR incubating time was depicted in Fig. 3A. The signal-to-noise ratio increased with the extension of incubating time. While the incubating time extended to 30 min, the signal generated by target DNA reached the largest and kept steady status. Hence, 30 min was chosen as the optimal reaction time. The reaction temperature was also examined from 4 to 48 °C (Fig. 3B), and the maximum signal-to-noise ratio were achieved at 37 °C. At low temperature, target DNA and complex QPR could not get a sufficient collision probability that significantly influenced the formation of double-stranded complex F-Q; at high temperature, the signal caused by non-stable complex QPR increased with the increase of incubating temperature. Thus, 37 °C was chosen as the optimal reaction temperature. In order to enhance detection sensitivity, the concentration of threestranded complex QPR and cruciform DNA crystal were also optimized. The effect of three-stranded complex QPR concentration on the signalto-noise ratio was investigated in Fig. 3C. With the augment of complex QPR concentration, the signal-to-noise ratio increased and then kept
3.4. Analytical performance of designed biosensor To evaluate the analytical performance of the established electrochemical biosensor, DPV responses to different concentrations of HIVrelated DNA were obtained under optimal conditions. As shown in Fig. 4A, The DPV peak current increased with the increase of target DNA concentration. The calibration plots showed a strong linear relationship between the peak currents and the logarithm of target DNA concentrations in the range from 1 pM to 100 nM (Fig. 4B). The resulting linear equation was ip (A) = 2.49 E−6 log CDNA (nM) + 1.05 E−5 with a correlation coefficient of 0.9978. According to the 3σ rule, the limit of detection (LOD) for target DNA was estimated to be 0.21 pM. For comparison, the analytical property of this method was compared with previously reported assays for nucleic acid detection. As shown in Table S2, the developed strategy had superior or comparable sensitivity to other nucleic acid detection strategies. The high sensitivity was attributed to the high efficiency of TSDR system and excellent signal amplification performance of cruciform DNA crystal. Hence, this
Fig. 2. (A) Native polyacrylamide gel electrophoresis results of the products and sequences of TSDR. Lane 1: 20 bp DNA ladder marker; Lane 2: HIV-related DNA; Lane 3: fuel strand F; Lane 4: three-stranded complex QPR; Lane 5: TSDR system without target DNA; Lane 6: TSDR system with target DNA. (B) Native polyacrylamide gel electrophoresis results of the products and sequences of cruciform DNA crystal. Lane 1: 20 bp DNA ladder marker; Lanes 2, 3, 4, 5: single strand a, b, c, d, respectively; Lane 6: annealing products of strand a and strand b; Lane 7: annealing products of strand a, strand b and strand c; Lane 8: cruciform DNA crystal.
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Fig. 3. Optimizations of experimental parameters: (A) evaluation of the effect of incubating time of TSDR, (B) evaluation of the effect of incubating temperature of TSDR, (C) evaluation of the effect of concentration of complex QPR, (D) evaluation of the effect of concentration of cruciform DNA crystal. The error bars represent the standard deviations calculated from three different spots.
biosensor can be applied to quantification of HIV-related DNA with a wide linear range and low detection concentration.
to that of the blank. However, in the presence of target DNA, there was a remarkable increase in the DPV current, which was about 5.5 times as that of SM and 11 times as that of DM and NC (Fig. 5B). These results indicated that the designed biosensor had remarkable specificity for the determination of HIV-related DNA. To estimate the repeatability of the developed biosensing strategy, the intra-assay precision of five different detections at one assay and inter-assay precision at five different assays for HIV-related DNA detection were measured, respectively. Consequently, the intra-assay coefficient of variation (CV) was 3.25% and the inter-assay CV was 4.43%, which manifested that this biosensor had an
3.5. Specificity and reproducibility of designed biosensor To evaluate the selectivity of the developed electrochemical method, four distinct control DNA sequences, including target DNA, single-base mismatch strand (SM), double-base mismatch strand (DM), and non-complementary sequence (NC) were used. As showed in Fig. 5A, DPV currents for 100 pM of DM and 100 pM of NC were similar
Fig. 4. (A) Typical DPV curves responding to 0, 0.001, 0.01, 0.1, 1, 10, and 100 nM of target DNA (from a to g). (B) Plot of DPV peak current vs. logarithm of target DNA concentration. 70
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Fig. 5. (A) Typical DPV curves and (B) DPV peak currents respectively respond to 100 pM of HIV-related DNA (a), single-base mismatched oligonucleotides (b), twobase mismatched oligonucleotides (c), non-complementary oligonucleotides (d), and blank (e).
acceptable reproducibility.
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3.6. Interfering effects of complex matrix To evaluate the interfering effects of complicated matrix on the proposed biosensor, the recovery assays were performed for the detection of target DNA in 10-fold diluted human serum samples. With a standard addition method, different amounts of target DNA were spiked into human serum samples for the assay. The recoveries were 94–106% from 10 pM to 10 nM (Table S3), indicating acceptable analytic performance in complex matrix. These good results were attributed to the diluted serum samples and several washing and separation steps to remove the nonspecific interferents. It implied that this method can be used for quantification of HIV-related DNA in complex biological matrix. 4. Conclusion To conclude, a simple and enzyme-free electrochemical biosensing strategy has been developed for HIV-related DNA detection by TSDR and cruciform DNA crystal cascade amplification. The enzyme-free strategy overcomes the disadvantages of traditional enzyme-based amplification and obtained satisfactory results. This electrochemical biosensor shows high sensitivity for the detection of HIV-related DNA with the dynamic linear range from 1 pM to 100 nM and the detection limit down to 0.21 pM. In addition, the designed biosensor possesses the advantages of simple material, excellent specificity, good reproducibility, and has been applied to analyze target DNA in human serum samples. Thus, this electrochemical biosensor would provide a promising platform for the detection of HIV-related DNA in biomedical research and early clinical diagnosis of HIV infection. Acknowledgements This work was funded by the Foundation for Science and Technology Research Project of Chongqing (KJ1502703). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jelechem.2018.05.011. References [1] E. Girardi, C.A. Sabin, A.D. Monforte, Late diagnosis of HIV infection: epidemiological features, consequences and strategies to encourage earlier testing, J. Acquir.
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