Letter Cite This: Anal. Chem. XXXX, XXX, XXX−XXX
pubs.acs.org/ac
Bipedal DNA Walker Based Electrochemical Genosensing Strategy Hua Chai†,‡ and Peng Miao*,†,‡ †
Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou 215163, P. R. China University of Science and Technology of China, Hefei 230026, P. R. China
‡
Anal. Chem. Downloaded from pubs.acs.org by 91.243.91.159 on 04/03/19. For personal use only.
S Supporting Information *
ABSTRACT: DNAs are one of the most fundamental molecules for life. Quantification of specific sequences is of great importance for biological research and clinical diagnosis. In order to determine extremely low abundant DNAs, we herein develop a novel electrochemical genosensor taking advantage of a smart bipedal DNA walking machine. Magnetic nanomaterials are first employed to enrich target DNA. Strand displacement amplification initiated by target DNA is then designed on the surface of the nanomaterials, the products of which can be used to trigger bipedal DNA walking on the surface of an electrode. Benefiting from triple amplification, ultrahigh sensitivity is achieved for electrochemical analysis of DNA. More importantly, the proposed strategy opens a new avenue for employing the bipedal DNA walker for sensitive detection of various biomolecules with signal amplification.
I
exonuclease III-assisted target recycling and DNA walker for fluorescent detection of carcinoembryonic antigen (CEA) with a limit of detection as low as 1.2 pg mL−1.13 Peng et al. fabricated a miRNA-powered DNA walker based on an enzyme-free electrochemiluminescence (ECL) energy transfer strategy.14 Zhu et al. developed a pixel counting strategy for nucleic acids assay by employing DNA walker-triggered fluorescence spots.15 In this work, we have developed a bipedal DNA walker for electrochemical genosensing with triple amplification including magnetic enrichment, strand displacement amplification, and DNAzyme based DNA walking. The detailed working principle is illustrated in Scheme 1. To achieve convenient pretreatment of biological samples and enrichment of target DNA, magnetic Fe3O4@gold nanoparticles (Fe3O4@AuNPs) are synthesized and used. Generally, DNA probe A is immobilized on the surface of Fe3O4@AuNPs via gold−sulfur self-assembly.16 Target DNA is able to hybridize with probe A. Due to the high surface to volume ratio of the magnetic nanomaterials, the loaded probe A with a high density can be used to enrich target DNA from the samples to be tested after a facile magnetic separation. Moreover, strand displacement amplification can be conducted on the surface of Fe3O4@AuNPs. In the presence of polymerase and nicking endonuclease, target-induced recycled DNA polymerization, nicking, and strand displacement reactions occur;17 meanwhile, a large number of DNA sequences (probe B) are produced and released for subsequent DNA walking.
t is universally known that infectious diseases and cancers are serious threats to human health.1,2 Early diagnosis of these diseases is of great significance to achieve more effective management and higher survival chances.3 So far, different promising diagnostic biomarkers have been discovered including DNAs,4 RNAs,5 proteins,6 cells,7 and so on. Sequence-specific detection of DNA always plays a critical role in the disease diagnosis and treatment.8 At present, many techniques have been widely applied including polymerase chain reaction (PCR),9 surface enhanced Raman spectroscopy (SERS) sensor,10 mass spectrometry assay,11 and fluorescent assay.12 However, there are certain limitations of these method for practical applications. For example, PCR is not suitable for the analysis of short-length oligonucleotides; synthetic steps of most SERS probes are complicated; and fluorescent assays may require expensive reagents and instruments. In addition, the pretreatment of biological samples is always laborious and time-consuming. Hence, the development of reliable, convenient, and sensitive methods for DNA assay is still highly desired. Besides the role of genetic information carrier, DNA also belongs to a kind of biomaterials, which can be used as the architectural nanoscaffold for applications in the field of nanotechnology. Synthetic DNA walker is a typical dynamic DNA device, which moves along the DNA track and aids the transduction and amplification of signals with remarkable locomotion and controllability. Structurally, this kind of DNA machine is composed of at least three essential components, including a walker, a track with overhanging branches as footholds, and certain forms of energy input as the driving force. DNA walker has gained tremendous attention from analytical communities for the detection of nucleic acids, proteins, and cells. For example, He et al. combined © XXXX American Chemical Society
Received: March 3, 2019 Accepted: April 2, 2019 Published: April 2, 2019 A
DOI: 10.1021/acs.analchem.9b01118 Anal. Chem. XXXX, XXX, XXX−XXX
Letter
Analytical Chemistry
on the surface of Fe3O4NPs with even distribution (Figure S1). The synthesized AgNPs are characterized by transmission electron microscopy (TEM). The spherical nanoparticles are well dispersed in water and the diameter centers at 4 nm (Figure S2). Polyacrylamide gel electrophoresis experiments are then performed to characterize DNA reactions. Figure 1A depicts
Scheme 1. Illustration of the Bipedal DNA Walker Based Electrochemical Genosensor
Different from traditional DNA walking machines, this proposed DNA walker device is composed of probe A modified Fe3O4@AuNPs, polymerase/nicking endonuclease for strand displacement amplification, tetrahedral DNA (TDNA) supported track, and two proximity probes as bipedal DNA walker sequences. First, TDNA nanostructured scaffold modified on the gold electrode not only enhances molecular recognition efficiency of the supported DNA track but also avoids spacer molecules for electrode surface modulation. With the 5′ labeled amino group of the DNA track, silver nanoparticles (AgNPs) can be localized on the interface of the electrode via the chemical bond formed between silver and amino nitrogen.18 Due to the highly characteristic solid-state Ag/AgCl reaction, AgNPs provide well-defined sharp silver stripping peaks, which are suitable to be used as excellent electrochemical signal sources.19 Second, the two proximity probes (probes C and D) are designed as two walkers, which contain the same metal ion-dependent DNAzyme tail sequence (Table S1). Although they are complementary with DNA track on top of TDNA, single probe C or D cannot associate with DNA track since the complementary fragments are short and the melting temperature is low. However, probe B generated by target DNA-induced strand displacement amplification is able to help the formation of a DNA star trigon nanostructure, and the tail sequences of probes C and D are brought into close proximity. Thus, the melting temperature for DNA star trigon nanostructure and DNA track is increased. After effective annealing, the DNAzyme cleaves the DNA track in the presence of Pb2+, resulting in the removal of the singlestranded DNA segment with the amino group. Meanwhile, the single dissociated DNA walker (probe C or D) explores neighboring intact substrate and the walking process is proceeded. The elimination of amino groups by DNA walking disables the adsorption of AgNPs as electrochemical nanoprobes. Therefore, by analyzing the electrochemical signals, ultrasensitive quantitative analysis of target DNA is achieved with triple signal amplification. The synthesized nanomaterials in this work are well characterized. As shown in the scanning electron microscopic (SEM) images, Fe3O4NPs show uniform spherical morphology with the diameter around 150 nm; after reduction of HAuCl4, small AuNPs (average diameter, 5 nm) are formed and coated
Figure 1. Polyacrylamide gel electrophoresis analysis of (A) the formation of TDNA: (a) probe Tb, (b) probes Tb and Tc, (c) probes Tb, Tc, and Td, (d) probes Ta, Tb, Tc, and Td. (B) Strand displacement polymerization: (a) probe A, (b) probe A and target, (c) probe A and target in the presence of Klenow fragment and Nb.BbvCI. (C) DNA walker: (a) probe C, (b) probe D, (c) probes C and D, (d) probes B, C, and D. (D) Cyclic voltammograms and (E) nyquist diagrams of bare electrode and TDNA modified electrode after incubation with probes C and D in the absence and presence of target DNA.
the sizes of the DNA structures formed by one, two, three, and four fuel strands of TDNA. By comparing the molecule weights, it can be clearly concluded that TDNA is successfully constructed with the four DNA probes. Next, strand displacement amplification is verified. As listed in Figure 1B, the band of probe A is clearly indicated in lane a. After hybridization with target DNA, a new band with larger molecule weight appears, which is ascribed to the formed double-stranded DNA (lane b). In the presence of Klenow fragment polymerase and Nb.BbvCI nicking endonuclease, polymerization and nicking reactions occur, generating a complete double-stranded DNA and multiple displaced probe B molecules, which are confirmed by lane c. The DNA products of conjugation reaction of proximity probes in the absence and presence of probe B are also compared in the gel image (Figure 1C). The molecule weights of probes C and D are similar. Since they cannot associate with each other without probe B, the band position of the mixture of probes C and D are nearly the same with probe C or D (lane c). In the presence of probe B, a star trigon structured DNA is formed, which is reflected by the band with much larger molecular weight (lane d). In addition, DNAzyme cleavage reaction is also characterized. Only in the presence of the star trigon structured DNA, the DNA track can be cleaved, which is evidenced by the produced DNA band with smaller molecular B
DOI: 10.1021/acs.analchem.9b01118 Anal. Chem. XXXX, XXX, XXX−XXX
Letter
Analytical Chemistry
target triggers the production of probe B, which promote the DNA walking machine. Amino groups are then kept away from the electrode, which no longer has the ability to localize AgNPs. Therefore, the LSV peak decreases remarkably. The results demonstrate that it is suitable for quantitative analysis of target DNA by analyzing the peak currents of LSV curves. To achieve the best analytical performance of the genosensor, several key experimental conditions should be optimized. The electrochemical signal originates from AgNPs modified on TDNA. Therefore, we have prepared TDNA with a series of concentrations for electrode immobilization before incubation with AgNPs. With the increase of the concentration, a larger stripping peak is obtained (Figure S4). After comparison, a plateau is reached after the concentration is larger than 1 μM. Therefore, we have chosen 1 μM as the optimal value for subsequent experiments. Strand displacement amplification on Fe3O4@AuNPs and the DNA walking process are essential for the improvement of the sensitivity. However, shorter reaction times are important for practical application. We have then studied the effect of reaction times on final obtained peak current variations. Experimental results show that 1 h for strand displacement amplification and 2 h for DNA walking are enough to achieve the highest electrochemical responses (Figure S5A,B). The amplification efficiency of this DNAzyme based DNA walker relies on both the concentration ratio between walker and track and Pb2+ level. Since the density of track on the electrode surface is fixed, we have tested the concentrations of walkers (probes C and D) and Pb2+ for concentration optimizations. With the increases of the walkers concentration (from 1 to 50 nM) and Pb2+ concentration (from 1 to 20 μM), the electrochemical responses increase correspondingly and barely change with higher concentrations (Figure S5C,D). Therefore, 50 nM of walkers and 20 μM of Pb2+ are selected as optimal conditions for the following quantitative experiments. In order to explore the quantitative accuracy of the sensing strategy, we have studied the LSV performances of the biosensor for the detection of different amounts of target DNA under optimal conditions. As shown in Figure 3A, with the
weight (Figure S3). All gel results demonstrate successful DNA reactions. Fabrication of the electrochemical genosensor is verified by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and linear sweep voltammetry (LSV), respectively. As shown in Figure 1D, a pair of well-defined current peaks is observed for bare gold electrode. Since negatively charged DNA is able to repel [Fe(CN)6]3−/4−, the peaks decrease after modification with TDNA. Probes B and C cannot directly hybridize with TDNA; thus, the peaks barely change after the incubation with the two proximity probes. However, in the presence of target DNA, a number of probe B molecules are produced to promote the proximity of probes C and D, which facilitate the association of the DNAzyme sequences with the DNA track on top of TDNA. The subsequent cleavage reaction cycles make the electrode surface less negative. Thus, the intensities of the CV peaks recover to certain extents. EIS results are then recorded and summarized in Figure 1E. Generally, in an impedance spectrum, the size of the semicircle domain always indicates the electron transfer limited process. Bare gold electrode shows a straight line, indicating excellent electron transfer rate of the substrate electrode. After modification with TDNA, a large semicircle domain is observed. The diameter increases slightly after incubation with probes C and D, which is ascribed to nonspecific adsorption. However, after target-induced walking, the diameter decreases, demonstrating successful cleavage reactions. The EIS results are well consistent with those of CV. It has been reported that AgNPs can generate significant signals in LSV.20 In this work, we have employed AgNPs for quantitative analysis of target DNA. To test the feasibility, LSV responses of the electrode with different modifications have first been compared in Figure 2. Bare electrode cannot absorb
Figure 2. Linear sweep voltammograms of bare electrode and TDNA modified electrode after incubation with probes C and D in the absence and presence of target DNA. All electrodes are treated with AgNPs before measurement.
Figure 3. (A) Linear sweep voltammograms for the detection of DNA with the concentration of 1 fM, 2 fM, 5 fM, 10 fM, 20 fM, 100 fM, 1 pM, and 10 pM (from left to right). Electrochemical species: 0.1 M KCl. Scan rate: 100 mV s−1. (B) Calibration plot of the LSV peak current variation versus the logarithmic concentration of target DNA. Inset shows the linear range from 1 fM to 100 fM.
AgNPs, thus no LSV peak can be observed. After modification with TDNA, the amino groups on top facilitate the interaction with AgNPs. As a result, a sharp silver stripping peak appears. Since probes C and D cannot be associated with DNA track in the absence of probe B, no DNAzyme cleavage reactions occur. Consequently, the LSV peak barely changes. However, the
increase of target DNA, more probe B molecules are produced for the formation of bipedal DNA walker. Thus, less amino groups are left to absorb AgNPs and the LSV peak drops gradually. Detailed relationship between the logarithm of DNA concentration and the variation of peak current is depicted in Figure 3B. A good linear range is from 1 fM to 100 fM with the regression equation as follows: C
DOI: 10.1021/acs.analchem.9b01118 Anal. Chem. XXXX, XXX, XXX−XXX
Letter
Analytical Chemistry y = 21.560 + 326.224x
(R2 = 0.996, n = 4)
DNA can be enriched by the employment of Fe3O4@AuNPs with a facile magnetic separation process. The nanomaterials are also beneficial for the practical application in biological samples. Second, besides target enrichment, two additional amplification process are involved including strand displacement amplification and DNAzyme based DNA walking. Therefore, ultrahigh sensitivity is achieved with a low LOD of 0.22 fM. Third, different from traditional DNA walkers, this DNA nanomachine achieves bipedal DNA walking by taking advantage of the principle of proximity-dependent DNA hybridization. The two proximity legs cannot function without probe B produced by target DNA. Therefore, high selectivity can be promised. The genosensing strategy provides a potential candidate for the detection of trace DNA for the purposes of clinical diagnosis. In addition, it could also be extended for the analysis of various targets like proteins and cells by changing the molecule recognition elements into certain DNA probes.
in which y stands for the decreased peak current, and x is the logarithm of DNA concentration. The limit of detection (LOD) is calculated to be 0.22 fM based on the criterion of a signal-to-noise ratio of 3.21 After comparing with other existing DNA assays, this method shows higher sensitivity, which may be more suitable for the analysis of trace DNA (Table S2). The reproducibility of this electrochemical method is investigated by two interassays (the same electrode toward DNA assay using different sets of Fe3O4@AuNPs and four independent electrodes for DNA assay). After challenging different levels of target DNA, the average relative standard deviations of these assays are 3.40% and 1.94%, which are acceptable (Figure S6). Excellent reproducibility is thus demonstrated. We have further checked the stability of this method. TDNA modified electrodes have been stored in 10 mM Tris-HCl buffer (10 mM TCEP, pH 8.0) at 4 °C for 4 weeks. Afterward, the electrodes were used for the detection of target DNA with a concentration of 100 fM. The obtained signal intensities are over 94% of original values, confirming good stability of this electrochemical system. The selectivity of this method is investigated by introducing various mismatch DNAs. Figure 4 presents that all pure
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.9b01118. Experimental section; SEM and TEM images of nanomaterials; gel characterization of DNAzyme cleavage reaction; optimization of concentrations of TDNA, probe C, probe D, and Pb2+ and reaction times of strand displacement amplification and DNAzyme cleavage; reproducibility investigation; DNA sequences used in this study; comparison of analytical performances of recent DNA assays; and results challenging clinical serum samples (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*Phone: +86-512-69588279. E-mail:
[email protected]. ORCID
Peng Miao: 0000-0003-3993-4778 Author Contributions
All authors have given approval to the final version of the manuscript.
Figure 4. Comparison of LSV peak current variations for the detection of mismatch DNAs with and without the addition of target DNA. The concentrations are 1 pM.
Notes
The authors declare no competing financial interest.
■ ■
ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (Grant 81771929).
mismatch DNAs result in negligible variations of peak currents. Only in the presence of target DNA, significant peak decreases can be observed. These results confirm the developed method is highly selective toward the detection of target DNA. To further explore the practical applicability of this method, clinical serum samples have been challenged. After spiked with different amount of target DNA, the serum samples are measured by the electrochemical biosensor. The results are listed in Table S3, which are consistent with those of droplet digital PCR (ddPCR). The recoveries are satisfactory, and the relative standard deviations are no more than 5%, revealing that the proposed method is feasible and applicable for future applications in biological samples. In summary, an ultrasensitive electrochemical genosensor is developed combining bipedal DNA walker and strand displacement amplification on magnetic nanomaterials. The main features and advantages are listed as follows. First, target
REFERENCES
(1) Cahn, P.; Madero, J. S.; Arribas, J. R.; Antinori, A.; Ortiz, R.; Clarke, A. E.; Hung, C. C.; Rockstroh, J. K.; Girard, P. M.; Sievers, J.; Man, C.; Currie, A.; Underwood, M.; Tenorio, A. R.; Pappa, K.; Wynne, B.; Fettiplace, A.; Gartland, M.; Aboud, M.; Smith, K.; GEMINI-Study-Team. Lancet 2019, 393, 143−155. (2) Ciccarelli, F. D. Nature 2019, 565, 301−303. (3) Kapur, S.; Barbhaiya, C.; Deneke, T.; Michaud, G. F. Circulation 2017, 136, 1247−1255. (4) Das, J.; Ivanov, I.; Sargent, E. H.; Kelley, S. O. J. Am. Chem. Soc. 2016, 138, 11009−11016. (5) Jeck, W. R.; Sharpless, N. E. Nat. Biotechnol. 2014, 32, 453−461. (6) Xu, S. T.; Ma, W.; Bai, Y.; Liu, H. W. J. Am. Chem. Soc. 2019, 141, 72−75. D
DOI: 10.1021/acs.analchem.9b01118 Anal. Chem. XXXX, XXX, XXX−XXX
Letter
Analytical Chemistry (7) Ahmed, M. G.; Abate, M. F.; Song, Y. L.; Zhu, Z.; Yan, F.; Xu, Y.; Wang, X. M.; Li, Q. B.; Yang, C. Y. Angew. Chem., Int. Ed. 2017, 56, 10681−10685. (8) Das, J.; Ivanov, I.; Safaei, T. S.; Sargent, E. H.; Kelley, S. O. Angew. Chem., Int. Ed. 2018, 57, 3711−3716. (9) Byrnes, S. A.; Chang, T. C.; Huynh, T.; Astashkina, A.; Weigl, B. H.; Nichols, K. P. Anal. Chem. 2018, 90, 9374−9380. (10) Su, J.; Wang, D. F.; Norbel, L.; Shen, J. L.; Zhao, Z. H.; Dou, Y. Z.; Peng, T. H.; Shi, J. Y.; Mathur, S.; Fan, C. H.; Song, S. P. Anal. Chem. 2017, 89, 2531−2538. (11) Guo, C.; Xie, C.; Chen, Q.; Cao, X. J.; Guo, M. Z.; Zheng, S.; Wang, Y. S. Anal. Chim. Acta 2018, 1034, 110−118. (12) Chen, Y. H.; Duong, H. T. T.; Wen, S. H.; Mi, C.; Zhou, Y. Z.; Shimoni, O.; Valenzuela, S. M.; Jin, D. Y. Anal. Chem. 2018, 90, 663− 668. (13) He, M. Q.; Wang, K.; Wang, W. J.; Yu, Y. L.; Wang, J. H. Anal. Chem. 2017, 89, 9292−9298. (14) Peng, L. C.; Zhang, P.; Chai, Y. Q.; Yuan, R. Anal. Chem. 2017, 89, 5036−5042. (15) Zhu, L. Y.; Liu, Q. H.; Yang, B. Y.; Ju, H. X.; Lei, J. P. Anal. Chem. 2018, 90, 6357−6361. (16) Hu, L.; Kong, J. M.; Han, D. X.; Niu, L.; Zhang, X. J. ACS Sens. 2019, 4, 235−241. (17) Miao, P.; Jiang, Y. T.; Zhang, T.; Huang, Y.; Tang, Y. G. Chem. Commun. 2018, 54, 7366−7369. (18) Mandal, A.; Sekar, S.; Chandrasekaran, N.; Mukherjee, A.; Sastry, T. S. RSC Adv. 2015, 5, 15763−15771. (19) Miao, P.; Han, K.; Sun, H. X.; Yin, J.; Zhao, J.; Wang, B. D.; Tang, Y. G. ACS Appl. Mater. Interfaces 2014, 6, 8667−8672. (20) Wei, T. X.; Zhang, W. W.; Tan, Q.; Cui, X. W.; Dai, Z. H. Anal. Chem. 2018, 90, 13051−13058. (21) Currie, L. A. Anal. Chim. Acta 1999, 391, 127−134.
E
DOI: 10.1021/acs.analchem.9b01118 Anal. Chem. XXXX, XXX, XXX−XXX
