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Short-chain oligonucleotide detection by glass nanopore using targeting-induced DNA tetrahedron deformation as signal amplifier
Analytica Chimica Acta 1063 (2019) 57e63
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Short-chain oligonucleotide detection by glass nanopore using targeting-induced DNA tetrahedron deformation as signal amplifier Dandan Wang a, b, 1, Xiaolong Xu a, 1, Ya Zhou a, b, Haijuan Li a, Guohua Qi a, c, Ping Hu a, b, Yongdong Jin a, b, c, * a b c
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, China University of Science and Technology of China, Hefei, Anhui, 230026, China University of Chinese Academy of Sciences, Beijing, 100049, China
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This report employ a successful strategy to detect short-chain ssDNA by glass capillary nanopore with ~20 nm diameter. It mainly uses target-triggered shape transformation of DNA-tetrahedron nanostructures as a signal amplifier. This method is capable of specifically and quantitatively detecting short single-stranded DNA I0.
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Article history: Received 16 January 2019 Received in revised form 21 February 2019 Accepted 22 February 2019 Available online 11 March 2019
Glass capillary nanopore has been developed as a promising sensing platform for bioassay with singlemolecule resolution. Although the diameter of glass capillary nanopore can be easily tuned, direct eventreadouts of small biomacromolecules, like short-chain oligonucleotide fragments (within ~20 nucleotides) remain great challenge, which limited by the configuration of the conical-shaped nanopore and the instrumental temporal resolution. Here, we exploit a smart strategy for glass nanopore detection of short-chain oligonucleotides by using relatively big-sized tetrahedral DNA nanostructures as a signal amplifier, which can amplify the signals and retard the translocation speed meanwhile. The tetrahedral DNA nanostructure with a hairpin loop sequence in one edge, undergoes a shape transformation upon the complementary combination of the target oligonucleotides, in which the presence of short-chain target oligonucleotide can be readout due to obvious variation in amplitude of ion current pulse that caused by volume change of the DNA tetrahedral. Therefore, this strategy is promising for extending glass nanopore sensing platform for sensitive detection of short-chain oligonucleotides. © 2019 Elsevier B.V. All rights reserved.
Keywords: Glass capillary nanopore Short-chain oligonucleotides Tetrahedral DNA nanostructures Current pulse signals
1. Introduction * Corresponding author. State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China. E-mail address: [email protected] (Y. Jin). 1 Wang Dandan and Xu Xiaolong have contributed equally to the article https://doi.org/10.1016/j.aca.2019.02.058 0003-2670/© 2019 Elsevier B.V. All rights reserved.
Oligonucleotides are a class of short-chain nucleotides with typical length less than 20 bases, including short-chain singlestrand DNA (ssDNA) and short-chain RNA (microRNA and siRNA). In recent years, researches on oligonucleotides, especially on
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microRNAs, have attracted enormous attentions due to their important regulatory functions in many physiological processes, such as cell differentiation, proliferation and apoptosis, and are closely associated with various diseases, especially cancers [1]. Traditional methods for sensitive oligonucleotides detection are mainly limited in polymerase chain reaction (PCR) [2], fluorescence [3], and plasmonic sensing [4], but they all have their strengths and weaknesses in detection. Therefore, it is highly desired to develop simple, rapid and label-free methods for the detection of shortchain oligonucleotides. Recently, nanopore analytics have gained increasing attention as a powerful single-molecule sensing platform [5]. So far, several biological nanopores have been applied for oligonucleotide sensing analysis by obtaining sufficient current and temporal resolution [5e7]. Nevertheless, the deficiency in stability (for all protein nanopore-lipid bilayer systems) limits their use in harsh pH, temperature and ionic concentration conditions [8]. Meanwhile, artificial solid-state nanopores, including silicon nitride (SiNx) [9], graphene [10], and MoS2 [11], have made great progress in detecting short ssDNA. However, these artificial nanopores usually need sophisticated equipment and time-consuming preparations. Very recently, single glass capillary-based nanopore has also been developed into a powerful platform for biosensing on singlemolecule level and has gained increasing attention due to its advantages of rigidity, stability, low-cost and easy preparation with adjustable size and shape [8,12,13]. Although single glass nanopipette has been employed in varied fields, such as scanning electrochemical microscope/scanning ion conductance microscopy (SECM/SICM) [14,15], nanoelectrode [16e18] and single cell surgery [19], the use of glass capillary as a nanopore sensing platform for DNA sensing [20,21], especially for single-strand DNA (ssDNA) with short chain length down to ~ 20 nucleotides (nts) is still challenging. In this study, we attempt to extend the use of glass capillary nanopore for short-chain oligonucleotides detection. Although the diameter of glass nanopores could be tuned down to 10 nm, direct event-readouts of short-chain oligonucleotide fragments (within ~20 nts) remain a challenge due to the restriction of conical-shape of the nanopore and instrumental temporal resolution. However, this can be solved by indirect detection using (larger sized) structurally variable bio-tags as signal amplifiers as the variation of biomolecular structure or volume can be reflected in the
information of current amplitude and duration time when they translocating through a glass nanopore [22]. For instance, we have recently succeeded in using small glass nanopore to characterize in situ DNA organization together with its concatemer generated from hybridization chain reaction at the single-molecule level [12]. Very recently, Actis and coworkers reported a very similar work using glass nanopipettes for structural analysis of 2D DNA origami [23]. Herein, we demonstrate an effective strategy for glass nanopore detection of short-chain oligonucleotides by using relatively bigsized DNA-tetrahedron nanostructures as signal amplifier, which undergoes an obvious shape/volume transformation/change upon the complementary combination of target oligonucleotides. DNA tetrahedron nanostructures, originally developed by Turberfield and co-workers [24], have been recently actively explored for a wide range of applications, such as sensors [25,26], logic gates [27], fluorescent nanotags [28], and drug delivery [29,30]. In this study, we employ the DNA-tetrahedron nanostructure with one edge containing a hairpin loop sequence created by Fan's group [27] to act as signal amplifier for resistive-pulse sensing of short-chain oligonucleotides (I0, 20 nts) (Scheme 1). The DNA-tetrahedron nanostructure was synthesized according to the reported method [27] and characterized by 15% polyacrylamide gel electrophoresis (PAGE, Fig. S1). In principle, when target I0 is complementary to the sequence, the hairpin loop opens and the edge is stretched, resulting in a significant size increase of entire tetrahedron. As size (volume/ length) or size change of target molecule is a key issue for resistivepulse sensing [31], the increase of DNA-tetrahedron volume will result in an increased amplitude of current pulse in the translocation process, achieving single molecule detection of target I0. 2. Experimental 2.1. Chemical and reagents All oligonucleotides (Table S1) were synthesized and purified by Sangon Biotech (Shanghai, China). Tris-HCl (pH 8.0), 50 TAE, 5 TBE, ammonium persulfate (APS) and Trimethylethylenediamine (TEME) and 30% (w/v) acrylamide/methylene bis-acrylamide solution (29: 1) were purchased from Sangon Biotech. Synthesis reaction buffer for DNA tetrahedron was Tris buffer (20 mM, pH 8.0) containing 50 mM MgCl2; electrolyte for tetrahedrons translocation through nanopore was 20 mM Tris-HCl, containing 50 mM MgCl2,
Scheme 1. Schematic of glass nanopore detection of short-chain ssDNA (I0, 20 nts) by using targeting-induced DNA-tetrahedron deformation as signal amplifier. In the presence of target I0, the complementary incorporation of I0 to the hairpin loop increases size/volume of the DNA-tetrahedron, enhancing resistive-pulse signals.
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0.3 M NaCl, 5 mM KCl, pH 8.0. All solutions were prepared with Milli-Q water (18 MU cm resistivity) from a Millipore system. 2.2. Self-assembly of DNA tetrahedron structure The sequences of all oligonucleotides used in this study are shown in Table S1. DNA tetrahedra were synthesized by mixing equimolar strands (combinations of DNA strands were shown in Table S2) in the TM buffer (20 mM Tris, 50 mM MgCl2, pH 8.0) with a final concentration of 1 mM. The mixture was heated to 95 C for 2 min and then cooled to 4 C in 30 s [27]. Target strand I0 was dissolved in buffer with a concentration of 100 mM, and it could be mixed with T-DT in different molar ratios by the above mentioned anneal protocol. All synthesized DNA tetrahedra were analyzed with 2% agarose gels at a constant voltage of 120 V for 1.5 h in the 1 TAE buffer (R-TD) or a 15% native polyacrylamide gel electrophoresis (PAGE) at a constant voltage of 120 V for 3 h in the 1 TBE buffer (T-DT and S-DT).
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concentration of 100 nM. The ionic currents were measured with Ag/AgCl electrodes inserted in buffer solution (in 20 mM Tris-HCl, containing 50 mM MgCl2, 0.3 M NaCl, 3 mM KCl, pH ¼ 8.0) in a homemade electrolyte cell and recorded with the amplifier Axopatch 200B (Molecular Devices) in voltage-clamp mode using a low-pass Bessel filter of 5 kHz. The signals were digitized with DigiData 1440A digitizer (Molecular Devices) at 100 kHz and viewed with Clampfit 10.2 software (Molecular Devices). The Ag/ AgCl electrode inserted in prepared quartz nanopipette filled with solution serves as working electrode, while another Ag/AgCl electrode was immersed in the solution outside the glass nanopore as reference electrode. The It curves were recorded by applying 400 mV voltage. All experiments were carried out at room temperature (~22 C). The transmission electron microscopy (TEM) images of the glass nanopores were carried out by using of a FEI TECNAI F20 EM with an accelerating voltage of 200 kV equipped with an energy dispersive spectrometer. The tip (~2 mm) of the nanopipette as TEM sample was cut off and carefully transferred it to a folding grid.
2.3. Fabrication of glass nanopores 3. Results and discussions Quartz glass capillaries (QF100-70-10, Sutter Instrument Co.), with outer diameter of 1.0 mm and inner diameter of 0.7 mm, were used for the experiments. All glass capillaries were thoroughly cleaned by immersing in freshly prepared piranha solution (3:1 98% H2SO4/30% H2O2) for ~2 h in order to remove organic impurities on the surface. Then, the capillaries were thoroughly rinsed with deionized water to neutral and were rinsed several times with ethanol, and vacuum dried at 80 C prior to use. The glass nanopores were then fabricated by using a CO2-laser based micropipette puller system (model P-2000, Sutter Instruments Co.) with a single line program containing the following parameters: Heat ¼ 760, Fil ¼ 4, Vel ¼ 29, Del ¼ 140, Pull ¼ 168. The tip diameters of the nanopipettes were about 20 nm. 2.4. Characterizations We studied the ionic current change during DNA tetrahedron translocating through the nanopore with the DNA tetrahedron
The DNA tetrahedron used here has a tense-state held by the complementary stem of the hairpin initially, and upon incorporation of the complementary strand (target I0) the hairpin stem would turn stretched. During the transformation of DNAtetrahedron from tense-state (T-DT) to stretched-state (S-DT), the occupied volume of DNA-tetrahedron increases from ~23 nm3 to 32 nm3 [25],with ~40% increasement. Compared with ~1.1 nm diameter of the target I0, the current pulse signal of the S-DT would be increased more than 10 times in theory. On the other hand, the translocation of free 20-nts ssDNA typically occurs too rapidly to be resolved by resistive-pulse sensing [32,33]. After complementary with DNA-tetrahedron, the translocation of the I0 system would slow down to be detected. Fig. 1a&b show typical current traces of the DNA-tetrahedron translocation through a nanopore with orifice of ~20 nm (Fig. S2), before and after the introduction of target I0, the downward blockade at 400 mV in each current trace represents a translocation
Fig. 1. Current versus time traces of (a) T-DT and (b) S-DT translocating through a glass nanopore with orifice of ~20 nm, at an applied voltage of 400 mV. (c) The current trace without T-DT and S-DT for comparison. (dee) Contour plots of jDI/Ij distribution produced by T-DT and S-DT, respectively. (f) Superposition of the histogram of jDI/Ij distribution of T-DT and S-DT, and percentage represents the proportion of translocation events in each distribution interval of jDI/Ij accounted for the total translocation events.
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event of DNA. Obviously, the amplitude of the current change caused by S-DT is significantly larger than that of T-DTs. For comparison, Fig. 1c shows the current trace without T-DT and S-DT and only with I0. There are no current blockade signals in this case, which explains that the larger current pulse signals were generated by DNA tetrahedrons. Fig. 1d and e show the corresponding contour plots of jDI/Ij (DI represents the current blockade and I indicates the baseline current) versus dwell time. The characteristic current spikes can be classified into two categories, a lower one and a higher one, with jDI/Ij centered at 0.08 and 0.4, or 0.08 and 0.45, respectively, for T-DT and S-DT. By comparing the current profile with that of another regular DNA-tetrahedron nanostructure (RDT) having similar size to the one used here and with stretched state (Fig. S3), larger current signals are found when R-DTs translocate through the nanopore. Thus we infer that the higher jDI/Ij at 0.45 is attributed to the S-DT, and the lower one would be ascribed to the T-DT. Both DNA-tetrahedron nanostructures have peaks at jDI/Ij ¼ 0.08, which we ascribe it to an incomplete assembly of four single DNA strands that make up the DNA-tetrahedrons. In the 15%
native polyacrylamide gel electrophoresis image (Fig. S1), it appears bands of the hybridization of three DNA single strands (B, C and D), and it can confirm the successful assembly of the DNAtetrahedrons. The corresponding current trace versus dwell time is recorded at 400 mV (Fig. S4a). As shown in the contour plot (Fig. S4b) and jDI/Ij distribution histogram (Fig. S4c), there is a current peak at jDI/Ij ¼ 0.06, which implies that the jDI/Ij signals at 0.08 of T-DT and S-DT are due to the self-assembly of three singlestranded DNA (B, C and D). Fig. 1f demonstrates the histogram superposition of jDI/Ij distribution of T-DT and S-DT, exhibiting the Gaussian distribution, and the percentage represents the proportion of translocation events in each distribution interval of jDI/Ij accounting for the total translocation events. As seen from the statistical results, the jDI/Ij distribution peak position of S-DT has apparently right-shifted compared to T-DT. Therefore, the comparative translocation behaviors of these two DNAtetrahedrons confirm the success of the tactic for ultrasensitive nanopore detection of short-chain ssDNA down to ~20 nts. As seen from the Gaussian distribution overlay of jDI/Ij of the two DNA-
Fig. 2. Contour plots produced by S-DT with varied assemble ratio of T-DT to I0: (a)1: 0, (b)1: 1, (c)1: 2, (d)1: 5, (e)1: 10, (f)1:20, with constant concentration of T-DT (100 nM). (g) Superposition of jDI/Ij distribution of the above six ratios, and the percentage of jDI/Ij0.36 reflected the amount of S-DT. (h) The relationship of I0: T-DT with the S-DT percentage.
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tetrahedrons, although their volumes are different, the distribution of their jDI/Ij is partially overlapped, this is because of the anisotropic shape of the two DNA tetrahedral structures (Fig. S5). That is, when translocating through the nanopore sensing zone, the orientation of the DNA-tetrahedron (face, edge, or vertex) affects resulting signals. As seen from Fig. 1e, the distributions of the conductance (jDI/Ij) when DNA tetrahedral nanostructures translocating through the glass nanopore are slightly wide, this is caused by the some flexibility of DNA nanostructures [34]. It is worth noting that jDI/Ij relates with the ratio of cross-sectional area of the analyte to the opening area of nanopore (jDI/Ij ¼ SD/SN). For l-DNA, its jDI/Ij distribution peak is only 0.03 (Fig. S6), While jDI/Ij of the DNA-tetrahedron is about 13e15 times larger than that of l-DNA. This is consistent with the ratio of their cross-sectional area in translocation, which confirms the feasibility and success of the tactic for glass nanopore detection of short-chain ssDNA.
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Next, we investigate the effect of different ratio of T-DT to target I0 (T-DT: I0 ¼ 1: 0, 1: 1, 1: 2, 1: 5, 1: 10, 1: 20) on resistive-pulse signals, with the concentration of T-DT maintained constant (100 nM). As shown in Fig. 2(aef), the current spikes can also be classified mainly into two categories, and upon addition of I0 to TDT, jDI/Ij increases from ~0.32 to 0.40, which further confirm that the higher jDI/Ij centered at 0.40 is attributable to the S-DT state, while the lower one is ascribed to the T-DT state. The percentage of jDI/Ij distribution is shown in Fig. 2g. The proportion of jDI/I 0.36 in the total translocation events represents the amount of S-DT assembled. As the concentration of I0 increases, the percentage of jDI/Ij0.36 in the total translocation events increases, that is, the amount of S-DT increases. Fig. 2h shows the exponential relationship between the ratio of I0 to T-DT and the amount of S-DT. This is reasonable because when the amount of I0 increases to a certain value, it will be in a saturated state, and then the amount of S-DT
Figure 3. (a) 15% native polyacrylamide gel electrophoresis of T-DT formation and its combination with I0 and base-mismatched ssDNAs. (bef) Contour plots of jDI/Ij distribution of the translocation with assemblies when adding base-mismatched ssDNAs to 100 nM T-DT: (b) T-DT, (c) single-base mismatched Is, (d) double-bases mismatched Id, (e) completely mismatched In and (f) S-DT. (g) Their corresponding jDI/Ij distribution superposition diagrams.
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will increase slowly and finally reach a saturation. The reproducibility of the sensing platform is relatively good. For consistency, each figure in the manuscript is obtained using the same glass nanopore. Experimentally, although there are some differences/ fluctuations among different glass nanopores in ion current, it can be ensured that all the data obtained can distinguish well the two DNA tetrahedral structures. To further check selectivity of the method for practical use, we select three control ssDNAs instead of target I0 whose length is the same as that of I0 (20 nts), but with a single base mismatch (Is), double base mismatch (Id) and complete mismatch (In), respectively, to the hairpin loop sequence. As seen from Fig. 3a, the electrophoretic bands after adding the base-mismatched ssDNAs to T-DT are the same as that of DNA tetrahedron itself. This implies that the base-mismatched short ssDNAs cannot be assembled into the DNA-tetrahedron to form S-DT. Their translocation events are also recorded and counted as contour plots (Fig. 3b ~ f), and the distribution in percentage of jDI/Ij is statistically analyzed (Fig. 3g). By comparison with the S-TD, there are almost no or fewer translocation events at jDI/Ij 0.28 after adding the base-mismatched ssDNAs to T-DT, which is the same as that of T-DT itself. So that, by adding ssDNA that is base-mismatched with the hairpin loop sequence of T-DT, the hairpin loop will not be stretched due to failure in assembly into the S-DT state. Therefore, we conclude that the hairpin loop sequence of T-DT can be designed for specific detection of target I0, by using conical glass nanopore as a sensing platform. 4. Conclusions In this study, we report a successful strategy to detect shortchain ssDNA by glass capillary nanopore with ~20 nm diameter, using target-triggered shape transformation of DNA-tetrahedron nanostructures as a signal amplifier. Sensitive and selective nanopore detection of short-chain oligonucleotides down to ~20 nts is achieved by the tactic. By rational design of the nucleic acid sequences that make up the DNA-tetrahedron, the strategy is promising for extending glass nanopore sensing platform for sensitive detection of other short-chain oligonucleotides of interest. Although the selectivity is achieved, the detection sensitivity in the current study is not high enough as compared with reported other methods. The reason may be that although the amount of four single-stranded DNAs used to assemble DNA tetrahedral nanostructure is relatively large, the DNA tetrahedron assembly efficiency is not as high as expected, which reduces the sensitivity of detection. Further investigations are needed to solve this problem. Compared to biological and 2D artificial nanopores, one obvious advantage of the conical glass nanopore is that it can be extend for single cell surgery and potential detection of short-chain DNA and microRNA in individual cells. Declaration of interest statement We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. Acknowledgement This work was supported by the National Key Research and Development Program of China (Grant No. 2016YFA0201300), the National Natural Science Foundation of China (grant Nos. 21475125
and 21675146), the Instrument Developing Project of the Chinese Academy of Sciences (Grant No. YZ201666), and the Science and Technology Development Program of Jilin Province (Grant No. 20180520142JH). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2019.02.058. References [1] G.A. Calin, C.M. Croce, MicroRNA signatures in human cancers, Nat. Rev. Canc. 6 (2006) 857. [2] J. Dong, G. Chen, W. Wang, X. Huang, H. Peng, Q. Pu, F. Du, X. Cui, Y. Deng, Z. Tang, Colorimetric PCR-based microRNA detection method based on small organic dye and single enzyme, Anal. Chem. 9 (2018) 7107e7111. [3] Z. Jin, D. Geißler, X. Qiu, K.D. Wegner, N. Hildebrandt, A rapid, amplificationfree, and sensitive diagnostic assay for single-step multiplexed fluorescence detection of MicroRNA, Angew. Chem. Int. Ed. 54 (2015) 10024e10029. [4] G.K. Joshi, S. Deitz-McElyea, M. Johnson, S. Mali, M. Korc, R. Sardar, Highly specific plasmonic biosensors for ultrasensitive MicroRNA detection in plasma from pancreatic cancer patients, Nano Lett. 14 (2014) 6955e6963. [5] S. Howorka, Z. Siwy, Nanopore analytics: sensing of single molecules, Chem. Soc. Rev. 38 (2009) 2360e2384. [6] C. Cao, Y.-L. Ying, Z.-L. Hu, D.-F. Liao, H. Tian, Y.-T. Long, Discrimination of oligonucleotides of different lengths with a wild-type aerolysin nanopore, Nat. Nanotechnol. 11 (2016) 713e718. [7] X. Zhang, Y. Wang, B.L. Fricke, L.-Q. Gu, Programming nanopore ion flow for encoded multiplex MicroRNA detection, ACS Nano 8 (2014) 3444e3450. [8] W. Shi, A.K. Friedman, L.A. Baker, Nanopore Sensing, Anal. Chem. 89 (2017) 157e188. [9] M. Wanunu, T. Dadosh, V. Ray, J. Jin, L. McReynolds, M. Drndi c, Rapid electronic detection of probe-specific microRNAs using thin nanopore sensors, Nat. Nanotechnol. 5 (2010) 807. [10] C.A. Merchant, K. Healy, M. Wanunu, V. Ray, N. Peterman, J. Bartel, M.D. Fischbein, K. Venta, Z. Luo, A.T.C. Johnson, M. Drndi c, DNA translocation through graphene nanopores, Nano Lett. 10 (2010) 2915e2921. [11] K. Liu, J. Feng, A. Kis, A. Radenovic, Atomically thin molybdenum disulfide nanopores with high sensitivity for DNA translocation, ACS Nano 8 (2014) 2504e2511. [12] Z.T. Zhu, Y. Zhou, X.L. Xu, R. Wu, Y.D. Jin, B.L. Li, Adaption of a solid-state nanopore to homogeneous DNA organization verification and label-free molecular analysis without covalent modification, Anal. Chem. 90 (2018) 814e820. [13] X.L. Xu, C.P. Li, Y. Zhou, Y.D. Jin, Controllable shrinking of glass capillary nanopores down to sub-10 nm by wet-chemical silanization for signalenhanced DNA translocation, ACS Sens. 2 (2017) 1452e1457. [14] C.A. Morris, A.K. Friedman, L.A. Baker, Applications of nanopipettes in the analytical sciences, Analyst 135 (2010) 2190e2202. [15] M. Shen, R. Ishimatsu, J. Kim, S. Amemiya, Quantitative imaging of ion transport through single nanopores by high-resolution scanning electrochemical microscopy, J. Am. Chem. Soc. 134 (2012) 9856e9859. [16] S. Liu, Q. Li, Y. Shao, Electrochemistry at micro- and nanoscopic liquid/liquid interfaces, Chem. Soc. Rev. 40 (2011) 2236e2253. [17] H.S. White, A. Bund, Mechanism of electrostatic gating at conical glass nanopore electrodes, Langmuir 24 (2008) 12062e12067. [18] R. Gao, Y.L. Ying, Y.J. Li, Y.X. Hu, R.J. Yu, Y. Lin, Y.T. Long, A 30 nm nanopore electrode: facile fabrication and direct insights into the intrinsic feature of single nanoparticle collisions, Angew. Chem. Int. Ed. 57 (2018) 1011e1015. €l, J. Velmurugan, W. Nogala, M.V. Mirkin, C. Lu, M. Guille [19] Y. Wang, J.-M. Noe Collignon, F. Lemaître, C. Amatore, Nanoelectrodes for determination of reactive oxygen and nitrogen species inside murine macrophages, Proc. Natl. Acad. Sci. U.S.A. 109 (2012) 11534e11539. [20] M. Karhanek, J.T. Kemp, N. Pourmand, R.W. Davis, C.D. Webb, Single DNA molecule detection using nanopipettes and nanoparticles, Nano Lett. 5 (2005) 403e407. [21] X. Gong, A.V. Patil, A.P. Ivanov, Q. Kong, T. Gibb, F. Dogan, A.J. deMello, J.B. Edel, Label-free in-flow detection of single DNA molecules using glass nanopipettes, Anal. Chem. 86 (2014) 835e841. [22] L.J. Steinbock, S. Krishnan, R.D. Bulushev, S. Borgeaud, M. Blokesch, L. Feletti, A. Radenovic, Probing the size of proteins with glass nanopores, Nanoscale 6 (2014) 14380e14387. €lti, P. Actis, Analysis of 2D DNA origami with [23] M. Raveendran, A.J. Lee, C. Wa nanopipettes, ChemElectroChem 5 (2018) 3014e3020. [24] R.P. Goodman, I.A.T. Schaap, C.F. Tardin, C.M. Erben, R.M. Berry, C.F. Schmidt, A.J. Turberfield, Rapid chiral assembly of rigid DNA building blocks for molecular nanofabrication, Science 310 (2005) 1661e1665. [25] M. Lin, J. Wang, G. Zhou, J. Wang, N. Wu, J. Lu, J. Gao, X. Chen, J. Shi, X. Zuo, C. Fan, Programmable engineering of a biosensing interface with tetrahedral DNA nanostructures for ultrasensitive DNA detection, Angew. Chem. Int. Ed.
D. Wang et al. / Analytica Chimica Acta 1063 (2019) 57e63 54 (2015) 2151e2155. [26] H. Pei, N. Lu, Y. Wen, S. Song, Y. Liu, H. Yan, C. Fan, A DNA nanostructure-based biomolecular probe carrier platform for electrochemical biosensing, Adv. Mater. 22 (2010) 4754e4758. [27] H. Pei, L. Liang, G. Yao, J. Li, Q. Huang, C. Fan, Reconfigurable threedimensional DNA nanostructures for the construction of intracellular logic sensors, Angew. Chem. Int. Ed. 124 (2012) 9154e9158. € [28] H. Ozhalıcı-Ünal, B.A. Armitage, Fluorescent DNA nanotags based on a selfassembled DNA tetrahedron, ACS Nano 3 (2009) 425e433. [29] G. Zhou, M. Lin, P. Song, X. Chen, J. Chao, L. Wang, Q. Huang, W. Huang, C. Fan, X. Zuo, Multivalent capture and detection of cancer cells with DNA nanostructured biosensors and multibranched hybridization chain reaction amplification, Anal. Chem. 86 (2014) 7843e7848. [30] J. Li, H. Pei, B. Zhu, L. Liang, M. Wei, Y. He, N. Chen, D. Li, Q. Huang, C. Fan, Self-
[31] [32]
[33]
[34]
63
assembled multivalent DNA nanostructures for noninvasive intracellular delivery of immunostimulatory CpG oligonucleotides, ACS Nano 5 (2011) 8783e8789. W.-J. Lan, D.A. Holden, B. Zhang, H.S. White, Nanoparticle transport in conicalshaped nanopores, Anal. Chem. 83 (2011) 3840e3847. A.T. Carlsen, O.K. Zahid, J.A. Ruzicka, E.W. Taylor, A.R. Hall, Selective detection and quantification of modified DNA with solid-state nanopores, Nano Lett. 14 (2014) 5488e5492. C. Raillon, P. Granjon, M. Graf, L.J. Steinbock, A. Radenovic, Fast and automatic processing of multi-level events in nanopore translocation experiments, Nanoscale 4 (2012) 4916e4924. € pfrich, J.R. Burns, N. Fertig, U.F. Keyser, S. Howorka, BilayerA. Seifert, K. Go spanning DNA nanopores with voltage-switching between open and closed state, ACS Nano 9 (2015) 1117e1126.
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Short-chain oligonucleotide detection by glass nanopore using targeting-induced DNA tetrahedron deformation as signal amplifier Dandan Wang a, b, 1, Xiaolong Xu a, 1, Ya Zhou a, b, Haijuan Li a, Guohua Qi a, c, Ping Hu a, b, Yongdong Jin a, b, c, * a b c
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, China University of Science and Technology of China, Hefei, Anhui, 230026, China University of Chinese Academy of Sciences, Beijing, 100049, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
This report employ a successful strategy to detect short-chain ssDNA by glass capillary nanopore with ~20 nm diameter. It mainly uses target-triggered shape transformation of DNA-tetrahedron nanostructures as a signal amplifier. This method is capable of specifically and quantitatively detecting short single-stranded DNA I0.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 January 2019 Received in revised form 21 February 2019 Accepted 22 February 2019 Available online 11 March 2019
Glass capillary nanopore has been developed as a promising sensing platform for bioassay with singlemolecule resolution. Although the diameter of glass capillary nanopore can be easily tuned, direct eventreadouts of small biomacromolecules, like short-chain oligonucleotide fragments (within ~20 nucleotides) remain great challenge, which limited by the configuration of the conical-shaped nanopore and the instrumental temporal resolution. Here, we exploit a smart strategy for glass nanopore detection of short-chain oligonucleotides by using relatively big-sized tetrahedral DNA nanostructures as a signal amplifier, which can amplify the signals and retard the translocation speed meanwhile. The tetrahedral DNA nanostructure with a hairpin loop sequence in one edge, undergoes a shape transformation upon the complementary combination of the target oligonucleotides, in which the presence of short-chain target oligonucleotide can be readout due to obvious variation in amplitude of ion current pulse that caused by volume change of the DNA tetrahedral. Therefore, this strategy is promising for extending glass nanopore sensing platform for sensitive detection of short-chain oligonucleotides. © 2019 Elsevier B.V. All rights reserved.
Keywords: Glass capillary nanopore Short-chain oligonucleotides Tetrahedral DNA nanostructures Current pulse signals
1. Introduction * Corresponding author. State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China. E-mail address: [email protected] (Y. Jin). 1 Wang Dandan and Xu Xiaolong have contributed equally to the article https://doi.org/10.1016/j.aca.2019.02.058 0003-2670/© 2019 Elsevier B.V. All rights reserved.
Oligonucleotides are a class of short-chain nucleotides with typical length less than 20 bases, including short-chain singlestrand DNA (ssDNA) and short-chain RNA (microRNA and siRNA). In recent years, researches on oligonucleotides, especially on
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microRNAs, have attracted enormous attentions due to their important regulatory functions in many physiological processes, such as cell differentiation, proliferation and apoptosis, and are closely associated with various diseases, especially cancers [1]. Traditional methods for sensitive oligonucleotides detection are mainly limited in polymerase chain reaction (PCR) [2], fluorescence [3], and plasmonic sensing [4], but they all have their strengths and weaknesses in detection. Therefore, it is highly desired to develop simple, rapid and label-free methods for the detection of shortchain oligonucleotides. Recently, nanopore analytics have gained increasing attention as a powerful single-molecule sensing platform [5]. So far, several biological nanopores have been applied for oligonucleotide sensing analysis by obtaining sufficient current and temporal resolution [5e7]. Nevertheless, the deficiency in stability (for all protein nanopore-lipid bilayer systems) limits their use in harsh pH, temperature and ionic concentration conditions [8]. Meanwhile, artificial solid-state nanopores, including silicon nitride (SiNx) [9], graphene [10], and MoS2 [11], have made great progress in detecting short ssDNA. However, these artificial nanopores usually need sophisticated equipment and time-consuming preparations. Very recently, single glass capillary-based nanopore has also been developed into a powerful platform for biosensing on singlemolecule level and has gained increasing attention due to its advantages of rigidity, stability, low-cost and easy preparation with adjustable size and shape [8,12,13]. Although single glass nanopipette has been employed in varied fields, such as scanning electrochemical microscope/scanning ion conductance microscopy (SECM/SICM) [14,15], nanoelectrode [16e18] and single cell surgery [19], the use of glass capillary as a nanopore sensing platform for DNA sensing [20,21], especially for single-strand DNA (ssDNA) with short chain length down to ~ 20 nucleotides (nts) is still challenging. In this study, we attempt to extend the use of glass capillary nanopore for short-chain oligonucleotides detection. Although the diameter of glass nanopores could be tuned down to 10 nm, direct event-readouts of short-chain oligonucleotide fragments (within ~20 nts) remain a challenge due to the restriction of conical-shape of the nanopore and instrumental temporal resolution. However, this can be solved by indirect detection using (larger sized) structurally variable bio-tags as signal amplifiers as the variation of biomolecular structure or volume can be reflected in the
information of current amplitude and duration time when they translocating through a glass nanopore [22]. For instance, we have recently succeeded in using small glass nanopore to characterize in situ DNA organization together with its concatemer generated from hybridization chain reaction at the single-molecule level [12]. Very recently, Actis and coworkers reported a very similar work using glass nanopipettes for structural analysis of 2D DNA origami [23]. Herein, we demonstrate an effective strategy for glass nanopore detection of short-chain oligonucleotides by using relatively bigsized DNA-tetrahedron nanostructures as signal amplifier, which undergoes an obvious shape/volume transformation/change upon the complementary combination of target oligonucleotides. DNA tetrahedron nanostructures, originally developed by Turberfield and co-workers [24], have been recently actively explored for a wide range of applications, such as sensors [25,26], logic gates [27], fluorescent nanotags [28], and drug delivery [29,30]. In this study, we employ the DNA-tetrahedron nanostructure with one edge containing a hairpin loop sequence created by Fan's group [27] to act as signal amplifier for resistive-pulse sensing of short-chain oligonucleotides (I0, 20 nts) (Scheme 1). The DNA-tetrahedron nanostructure was synthesized according to the reported method [27] and characterized by 15% polyacrylamide gel electrophoresis (PAGE, Fig. S1). In principle, when target I0 is complementary to the sequence, the hairpin loop opens and the edge is stretched, resulting in a significant size increase of entire tetrahedron. As size (volume/ length) or size change of target molecule is a key issue for resistivepulse sensing [31], the increase of DNA-tetrahedron volume will result in an increased amplitude of current pulse in the translocation process, achieving single molecule detection of target I0. 2. Experimental 2.1. Chemical and reagents All oligonucleotides (Table S1) were synthesized and purified by Sangon Biotech (Shanghai, China). Tris-HCl (pH 8.0), 50 TAE, 5 TBE, ammonium persulfate (APS) and Trimethylethylenediamine (TEME) and 30% (w/v) acrylamide/methylene bis-acrylamide solution (29: 1) were purchased from Sangon Biotech. Synthesis reaction buffer for DNA tetrahedron was Tris buffer (20 mM, pH 8.0) containing 50 mM MgCl2; electrolyte for tetrahedrons translocation through nanopore was 20 mM Tris-HCl, containing 50 mM MgCl2,
Scheme 1. Schematic of glass nanopore detection of short-chain ssDNA (I0, 20 nts) by using targeting-induced DNA-tetrahedron deformation as signal amplifier. In the presence of target I0, the complementary incorporation of I0 to the hairpin loop increases size/volume of the DNA-tetrahedron, enhancing resistive-pulse signals.
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0.3 M NaCl, 5 mM KCl, pH 8.0. All solutions were prepared with Milli-Q water (18 MU cm resistivity) from a Millipore system. 2.2. Self-assembly of DNA tetrahedron structure The sequences of all oligonucleotides used in this study are shown in Table S1. DNA tetrahedra were synthesized by mixing equimolar strands (combinations of DNA strands were shown in Table S2) in the TM buffer (20 mM Tris, 50 mM MgCl2, pH 8.0) with a final concentration of 1 mM. The mixture was heated to 95 C for 2 min and then cooled to 4 C in 30 s [27]. Target strand I0 was dissolved in buffer with a concentration of 100 mM, and it could be mixed with T-DT in different molar ratios by the above mentioned anneal protocol. All synthesized DNA tetrahedra were analyzed with 2% agarose gels at a constant voltage of 120 V for 1.5 h in the 1 TAE buffer (R-TD) or a 15% native polyacrylamide gel electrophoresis (PAGE) at a constant voltage of 120 V for 3 h in the 1 TBE buffer (T-DT and S-DT).
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concentration of 100 nM. The ionic currents were measured with Ag/AgCl electrodes inserted in buffer solution (in 20 mM Tris-HCl, containing 50 mM MgCl2, 0.3 M NaCl, 3 mM KCl, pH ¼ 8.0) in a homemade electrolyte cell and recorded with the amplifier Axopatch 200B (Molecular Devices) in voltage-clamp mode using a low-pass Bessel filter of 5 kHz. The signals were digitized with DigiData 1440A digitizer (Molecular Devices) at 100 kHz and viewed with Clampfit 10.2 software (Molecular Devices). The Ag/ AgCl electrode inserted in prepared quartz nanopipette filled with solution serves as working electrode, while another Ag/AgCl electrode was immersed in the solution outside the glass nanopore as reference electrode. The It curves were recorded by applying 400 mV voltage. All experiments were carried out at room temperature (~22 C). The transmission electron microscopy (TEM) images of the glass nanopores were carried out by using of a FEI TECNAI F20 EM with an accelerating voltage of 200 kV equipped with an energy dispersive spectrometer. The tip (~2 mm) of the nanopipette as TEM sample was cut off and carefully transferred it to a folding grid.
2.3. Fabrication of glass nanopores 3. Results and discussions Quartz glass capillaries (QF100-70-10, Sutter Instrument Co.), with outer diameter of 1.0 mm and inner diameter of 0.7 mm, were used for the experiments. All glass capillaries were thoroughly cleaned by immersing in freshly prepared piranha solution (3:1 98% H2SO4/30% H2O2) for ~2 h in order to remove organic impurities on the surface. Then, the capillaries were thoroughly rinsed with deionized water to neutral and were rinsed several times with ethanol, and vacuum dried at 80 C prior to use. The glass nanopores were then fabricated by using a CO2-laser based micropipette puller system (model P-2000, Sutter Instruments Co.) with a single line program containing the following parameters: Heat ¼ 760, Fil ¼ 4, Vel ¼ 29, Del ¼ 140, Pull ¼ 168. The tip diameters of the nanopipettes were about 20 nm. 2.4. Characterizations We studied the ionic current change during DNA tetrahedron translocating through the nanopore with the DNA tetrahedron
The DNA tetrahedron used here has a tense-state held by the complementary stem of the hairpin initially, and upon incorporation of the complementary strand (target I0) the hairpin stem would turn stretched. During the transformation of DNAtetrahedron from tense-state (T-DT) to stretched-state (S-DT), the occupied volume of DNA-tetrahedron increases from ~23 nm3 to 32 nm3 [25],with ~40% increasement. Compared with ~1.1 nm diameter of the target I0, the current pulse signal of the S-DT would be increased more than 10 times in theory. On the other hand, the translocation of free 20-nts ssDNA typically occurs too rapidly to be resolved by resistive-pulse sensing [32,33]. After complementary with DNA-tetrahedron, the translocation of the I0 system would slow down to be detected. Fig. 1a&b show typical current traces of the DNA-tetrahedron translocation through a nanopore with orifice of ~20 nm (Fig. S2), before and after the introduction of target I0, the downward blockade at 400 mV in each current trace represents a translocation
Fig. 1. Current versus time traces of (a) T-DT and (b) S-DT translocating through a glass nanopore with orifice of ~20 nm, at an applied voltage of 400 mV. (c) The current trace without T-DT and S-DT for comparison. (dee) Contour plots of jDI/Ij distribution produced by T-DT and S-DT, respectively. (f) Superposition of the histogram of jDI/Ij distribution of T-DT and S-DT, and percentage represents the proportion of translocation events in each distribution interval of jDI/Ij accounted for the total translocation events.
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event of DNA. Obviously, the amplitude of the current change caused by S-DT is significantly larger than that of T-DTs. For comparison, Fig. 1c shows the current trace without T-DT and S-DT and only with I0. There are no current blockade signals in this case, which explains that the larger current pulse signals were generated by DNA tetrahedrons. Fig. 1d and e show the corresponding contour plots of jDI/Ij (DI represents the current blockade and I indicates the baseline current) versus dwell time. The characteristic current spikes can be classified into two categories, a lower one and a higher one, with jDI/Ij centered at 0.08 and 0.4, or 0.08 and 0.45, respectively, for T-DT and S-DT. By comparing the current profile with that of another regular DNA-tetrahedron nanostructure (RDT) having similar size to the one used here and with stretched state (Fig. S3), larger current signals are found when R-DTs translocate through the nanopore. Thus we infer that the higher jDI/Ij at 0.45 is attributed to the S-DT, and the lower one would be ascribed to the T-DT. Both DNA-tetrahedron nanostructures have peaks at jDI/Ij ¼ 0.08, which we ascribe it to an incomplete assembly of four single DNA strands that make up the DNA-tetrahedrons. In the 15%
native polyacrylamide gel electrophoresis image (Fig. S1), it appears bands of the hybridization of three DNA single strands (B, C and D), and it can confirm the successful assembly of the DNAtetrahedrons. The corresponding current trace versus dwell time is recorded at 400 mV (Fig. S4a). As shown in the contour plot (Fig. S4b) and jDI/Ij distribution histogram (Fig. S4c), there is a current peak at jDI/Ij ¼ 0.06, which implies that the jDI/Ij signals at 0.08 of T-DT and S-DT are due to the self-assembly of three singlestranded DNA (B, C and D). Fig. 1f demonstrates the histogram superposition of jDI/Ij distribution of T-DT and S-DT, exhibiting the Gaussian distribution, and the percentage represents the proportion of translocation events in each distribution interval of jDI/Ij accounting for the total translocation events. As seen from the statistical results, the jDI/Ij distribution peak position of S-DT has apparently right-shifted compared to T-DT. Therefore, the comparative translocation behaviors of these two DNAtetrahedrons confirm the success of the tactic for ultrasensitive nanopore detection of short-chain ssDNA down to ~20 nts. As seen from the Gaussian distribution overlay of jDI/Ij of the two DNA-
Fig. 2. Contour plots produced by S-DT with varied assemble ratio of T-DT to I0: (a)1: 0, (b)1: 1, (c)1: 2, (d)1: 5, (e)1: 10, (f)1:20, with constant concentration of T-DT (100 nM). (g) Superposition of jDI/Ij distribution of the above six ratios, and the percentage of jDI/Ij0.36 reflected the amount of S-DT. (h) The relationship of I0: T-DT with the S-DT percentage.
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tetrahedrons, although their volumes are different, the distribution of their jDI/Ij is partially overlapped, this is because of the anisotropic shape of the two DNA tetrahedral structures (Fig. S5). That is, when translocating through the nanopore sensing zone, the orientation of the DNA-tetrahedron (face, edge, or vertex) affects resulting signals. As seen from Fig. 1e, the distributions of the conductance (jDI/Ij) when DNA tetrahedral nanostructures translocating through the glass nanopore are slightly wide, this is caused by the some flexibility of DNA nanostructures [34]. It is worth noting that jDI/Ij relates with the ratio of cross-sectional area of the analyte to the opening area of nanopore (jDI/Ij ¼ SD/SN). For l-DNA, its jDI/Ij distribution peak is only 0.03 (Fig. S6), While jDI/Ij of the DNA-tetrahedron is about 13e15 times larger than that of l-DNA. This is consistent with the ratio of their cross-sectional area in translocation, which confirms the feasibility and success of the tactic for glass nanopore detection of short-chain ssDNA.
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Next, we investigate the effect of different ratio of T-DT to target I0 (T-DT: I0 ¼ 1: 0, 1: 1, 1: 2, 1: 5, 1: 10, 1: 20) on resistive-pulse signals, with the concentration of T-DT maintained constant (100 nM). As shown in Fig. 2(aef), the current spikes can also be classified mainly into two categories, and upon addition of I0 to TDT, jDI/Ij increases from ~0.32 to 0.40, which further confirm that the higher jDI/Ij centered at 0.40 is attributable to the S-DT state, while the lower one is ascribed to the T-DT state. The percentage of jDI/Ij distribution is shown in Fig. 2g. The proportion of jDI/I 0.36 in the total translocation events represents the amount of S-DT assembled. As the concentration of I0 increases, the percentage of jDI/Ij0.36 in the total translocation events increases, that is, the amount of S-DT increases. Fig. 2h shows the exponential relationship between the ratio of I0 to T-DT and the amount of S-DT. This is reasonable because when the amount of I0 increases to a certain value, it will be in a saturated state, and then the amount of S-DT
Figure 3. (a) 15% native polyacrylamide gel electrophoresis of T-DT formation and its combination with I0 and base-mismatched ssDNAs. (bef) Contour plots of jDI/Ij distribution of the translocation with assemblies when adding base-mismatched ssDNAs to 100 nM T-DT: (b) T-DT, (c) single-base mismatched Is, (d) double-bases mismatched Id, (e) completely mismatched In and (f) S-DT. (g) Their corresponding jDI/Ij distribution superposition diagrams.
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will increase slowly and finally reach a saturation. The reproducibility of the sensing platform is relatively good. For consistency, each figure in the manuscript is obtained using the same glass nanopore. Experimentally, although there are some differences/ fluctuations among different glass nanopores in ion current, it can be ensured that all the data obtained can distinguish well the two DNA tetrahedral structures. To further check selectivity of the method for practical use, we select three control ssDNAs instead of target I0 whose length is the same as that of I0 (20 nts), but with a single base mismatch (Is), double base mismatch (Id) and complete mismatch (In), respectively, to the hairpin loop sequence. As seen from Fig. 3a, the electrophoretic bands after adding the base-mismatched ssDNAs to T-DT are the same as that of DNA tetrahedron itself. This implies that the base-mismatched short ssDNAs cannot be assembled into the DNA-tetrahedron to form S-DT. Their translocation events are also recorded and counted as contour plots (Fig. 3b ~ f), and the distribution in percentage of jDI/Ij is statistically analyzed (Fig. 3g). By comparison with the S-TD, there are almost no or fewer translocation events at jDI/Ij 0.28 after adding the base-mismatched ssDNAs to T-DT, which is the same as that of T-DT itself. So that, by adding ssDNA that is base-mismatched with the hairpin loop sequence of T-DT, the hairpin loop will not be stretched due to failure in assembly into the S-DT state. Therefore, we conclude that the hairpin loop sequence of T-DT can be designed for specific detection of target I0, by using conical glass nanopore as a sensing platform. 4. Conclusions In this study, we report a successful strategy to detect shortchain ssDNA by glass capillary nanopore with ~20 nm diameter, using target-triggered shape transformation of DNA-tetrahedron nanostructures as a signal amplifier. Sensitive and selective nanopore detection of short-chain oligonucleotides down to ~20 nts is achieved by the tactic. By rational design of the nucleic acid sequences that make up the DNA-tetrahedron, the strategy is promising for extending glass nanopore sensing platform for sensitive detection of other short-chain oligonucleotides of interest. Although the selectivity is achieved, the detection sensitivity in the current study is not high enough as compared with reported other methods. The reason may be that although the amount of four single-stranded DNAs used to assemble DNA tetrahedral nanostructure is relatively large, the DNA tetrahedron assembly efficiency is not as high as expected, which reduces the sensitivity of detection. Further investigations are needed to solve this problem. Compared to biological and 2D artificial nanopores, one obvious advantage of the conical glass nanopore is that it can be extend for single cell surgery and potential detection of short-chain DNA and microRNA in individual cells. Declaration of interest statement We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. Acknowledgement This work was supported by the National Key Research and Development Program of China (Grant No. 2016YFA0201300), the National Natural Science Foundation of China (grant Nos. 21475125
and 21675146), the Instrument Developing Project of the Chinese Academy of Sciences (Grant No. YZ201666), and the Science and Technology Development Program of Jilin Province (Grant No. 20180520142JH). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2019.02.058. References [1] G.A. Calin, C.M. Croce, MicroRNA signatures in human cancers, Nat. Rev. Canc. 6 (2006) 857. [2] J. Dong, G. Chen, W. Wang, X. Huang, H. Peng, Q. Pu, F. Du, X. Cui, Y. Deng, Z. Tang, Colorimetric PCR-based microRNA detection method based on small organic dye and single enzyme, Anal. Chem. 9 (2018) 7107e7111. [3] Z. Jin, D. Geißler, X. Qiu, K.D. Wegner, N. Hildebrandt, A rapid, amplificationfree, and sensitive diagnostic assay for single-step multiplexed fluorescence detection of MicroRNA, Angew. Chem. Int. Ed. 54 (2015) 10024e10029. [4] G.K. Joshi, S. Deitz-McElyea, M. Johnson, S. Mali, M. Korc, R. Sardar, Highly specific plasmonic biosensors for ultrasensitive MicroRNA detection in plasma from pancreatic cancer patients, Nano Lett. 14 (2014) 6955e6963. [5] S. Howorka, Z. Siwy, Nanopore analytics: sensing of single molecules, Chem. Soc. Rev. 38 (2009) 2360e2384. [6] C. Cao, Y.-L. Ying, Z.-L. Hu, D.-F. Liao, H. Tian, Y.-T. Long, Discrimination of oligonucleotides of different lengths with a wild-type aerolysin nanopore, Nat. Nanotechnol. 11 (2016) 713e718. [7] X. Zhang, Y. Wang, B.L. Fricke, L.-Q. Gu, Programming nanopore ion flow for encoded multiplex MicroRNA detection, ACS Nano 8 (2014) 3444e3450. [8] W. Shi, A.K. Friedman, L.A. Baker, Nanopore Sensing, Anal. Chem. 89 (2017) 157e188. [9] M. Wanunu, T. Dadosh, V. Ray, J. Jin, L. McReynolds, M. Drndi c, Rapid electronic detection of probe-specific microRNAs using thin nanopore sensors, Nat. Nanotechnol. 5 (2010) 807. [10] C.A. Merchant, K. Healy, M. Wanunu, V. Ray, N. Peterman, J. Bartel, M.D. Fischbein, K. Venta, Z. Luo, A.T.C. Johnson, M. Drndi c, DNA translocation through graphene nanopores, Nano Lett. 10 (2010) 2915e2921. [11] K. Liu, J. Feng, A. Kis, A. Radenovic, Atomically thin molybdenum disulfide nanopores with high sensitivity for DNA translocation, ACS Nano 8 (2014) 2504e2511. [12] Z.T. Zhu, Y. Zhou, X.L. Xu, R. Wu, Y.D. Jin, B.L. Li, Adaption of a solid-state nanopore to homogeneous DNA organization verification and label-free molecular analysis without covalent modification, Anal. Chem. 90 (2018) 814e820. [13] X.L. Xu, C.P. Li, Y. Zhou, Y.D. Jin, Controllable shrinking of glass capillary nanopores down to sub-10 nm by wet-chemical silanization for signalenhanced DNA translocation, ACS Sens. 2 (2017) 1452e1457. [14] C.A. Morris, A.K. Friedman, L.A. Baker, Applications of nanopipettes in the analytical sciences, Analyst 135 (2010) 2190e2202. [15] M. Shen, R. Ishimatsu, J. Kim, S. Amemiya, Quantitative imaging of ion transport through single nanopores by high-resolution scanning electrochemical microscopy, J. Am. Chem. Soc. 134 (2012) 9856e9859. [16] S. Liu, Q. Li, Y. Shao, Electrochemistry at micro- and nanoscopic liquid/liquid interfaces, Chem. Soc. Rev. 40 (2011) 2236e2253. [17] H.S. White, A. Bund, Mechanism of electrostatic gating at conical glass nanopore electrodes, Langmuir 24 (2008) 12062e12067. [18] R. Gao, Y.L. Ying, Y.J. Li, Y.X. Hu, R.J. Yu, Y. Lin, Y.T. Long, A 30 nm nanopore electrode: facile fabrication and direct insights into the intrinsic feature of single nanoparticle collisions, Angew. Chem. Int. Ed. 57 (2018) 1011e1015. €l, J. Velmurugan, W. Nogala, M.V. Mirkin, C. Lu, M. Guille [19] Y. Wang, J.-M. Noe Collignon, F. Lemaître, C. Amatore, Nanoelectrodes for determination of reactive oxygen and nitrogen species inside murine macrophages, Proc. Natl. Acad. Sci. U.S.A. 109 (2012) 11534e11539. [20] M. Karhanek, J.T. Kemp, N. Pourmand, R.W. Davis, C.D. Webb, Single DNA molecule detection using nanopipettes and nanoparticles, Nano Lett. 5 (2005) 403e407. [21] X. Gong, A.V. Patil, A.P. Ivanov, Q. Kong, T. Gibb, F. Dogan, A.J. deMello, J.B. Edel, Label-free in-flow detection of single DNA molecules using glass nanopipettes, Anal. Chem. 86 (2014) 835e841. [22] L.J. Steinbock, S. Krishnan, R.D. Bulushev, S. Borgeaud, M. Blokesch, L. Feletti, A. Radenovic, Probing the size of proteins with glass nanopores, Nanoscale 6 (2014) 14380e14387. €lti, P. Actis, Analysis of 2D DNA origami with [23] M. Raveendran, A.J. Lee, C. Wa nanopipettes, ChemElectroChem 5 (2018) 3014e3020. [24] R.P. Goodman, I.A.T. Schaap, C.F. Tardin, C.M. Erben, R.M. Berry, C.F. Schmidt, A.J. Turberfield, Rapid chiral assembly of rigid DNA building blocks for molecular nanofabrication, Science 310 (2005) 1661e1665. [25] M. Lin, J. Wang, G. Zhou, J. Wang, N. Wu, J. Lu, J. Gao, X. Chen, J. Shi, X. Zuo, C. Fan, Programmable engineering of a biosensing interface with tetrahedral DNA nanostructures for ultrasensitive DNA detection, Angew. Chem. Int. Ed.
D. Wang et al. / Analytica Chimica Acta 1063 (2019) 57e63 54 (2015) 2151e2155. [26] H. Pei, N. Lu, Y. Wen, S. Song, Y. Liu, H. Yan, C. Fan, A DNA nanostructure-based biomolecular probe carrier platform for electrochemical biosensing, Adv. Mater. 22 (2010) 4754e4758. [27] H. Pei, L. Liang, G. Yao, J. Li, Q. Huang, C. Fan, Reconfigurable threedimensional DNA nanostructures for the construction of intracellular logic sensors, Angew. Chem. Int. Ed. 124 (2012) 9154e9158. € [28] H. Ozhalıcı-Ünal, B.A. Armitage, Fluorescent DNA nanotags based on a selfassembled DNA tetrahedron, ACS Nano 3 (2009) 425e433. [29] G. Zhou, M. Lin, P. Song, X. Chen, J. Chao, L. Wang, Q. Huang, W. Huang, C. Fan, X. Zuo, Multivalent capture and detection of cancer cells with DNA nanostructured biosensors and multibranched hybridization chain reaction amplification, Anal. Chem. 86 (2014) 7843e7848. [30] J. Li, H. Pei, B. Zhu, L. Liang, M. Wei, Y. He, N. Chen, D. Li, Q. Huang, C. Fan, Self-
[31] [32]
[33]
[34]
63
assembled multivalent DNA nanostructures for noninvasive intracellular delivery of immunostimulatory CpG oligonucleotides, ACS Nano 5 (2011) 8783e8789. W.-J. Lan, D.A. Holden, B. Zhang, H.S. White, Nanoparticle transport in conicalshaped nanopores, Anal. Chem. 83 (2011) 3840e3847. A.T. Carlsen, O.K. Zahid, J.A. Ruzicka, E.W. Taylor, A.R. Hall, Selective detection and quantification of modified DNA with solid-state nanopores, Nano Lett. 14 (2014) 5488e5492. C. Raillon, P. Granjon, M. Graf, L.J. Steinbock, A. Radenovic, Fast and automatic processing of multi-level events in nanopore translocation experiments, Nanoscale 4 (2012) 4916e4924. € pfrich, J.R. Burns, N. Fertig, U.F. Keyser, S. Howorka, BilayerA. Seifert, K. Go spanning DNA nanopores with voltage-switching between open and closed state, ACS Nano 9 (2015) 1117e1126.