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An efficient, label-free and sensitive electrochemical microRNA sensor based on target-initiated catalytic hairpin assembly of trivalent DNAzyme junctions
Sensors & Actuators: B. Chemical xxx (xxxx) xxxx
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
An efficient, label-free and sensitive electrochemical microRNA sensor based on target-initiated catalytic hairpin assembly of trivalent DNAzyme junctions Ronghui Ren, Qian Bi, Ruo Yuan, Yun Xiang
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Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
ARTICLE INFO
ABSTRACT
Keywords: Catalytic hairpin assembly Electrochemistry DNAzyme Biosensor MicroRNA
We have reported an efficient and ultrasensitive electrochemical sensor for label-free sensing of microRNAs (miRNAs) in serum samples via the formation of trivalent DNAzyme junctions through a target-initiated catalytic hairpin assembly (CHA) approach. The target miRNA sequences can lead to CHA of three hairpins into many trivalent DNAzyme junctions, which exhibit significantly enhanced efficiency over the conventional single DNAzyme structure for cleaving the G-quadruplex-containing DNA substrate sequences on the sensor surface. Such a cyclic and efficient cleavage thus results in the generation of massive free G-quadruplex sequences, which subsequently bind and confine the electroactive species, hemin, in the vicinity of the electrode for yielding drastically enhanced current intensity for ultrasensitive sensing of the target from 1 fM to 1 nM with a low detection limit of 0.46 fM. Besides, the sensor can discriminate single- and two-base mismatched sequences among the let-7 family with excellent selectivity and can be used to monitor the target let-7a in diluted serum sample, making such a sensor hold great potentials for highly sensitive and facile discrimination of other miRNA biomarkers.
1. Introduction MicroRNAs (miRNAs) with about 17 to 25 nucleotides in length are a kind of endogenous regulatory non-coding RNAs found in eukaryotes [1,2]. Evidences have shown that specifically expressed miRNAs are associated with many significant diseases and these specific miRNAs are used as potential biomarkers to diagnose these diseases [3–5]. It was subsequently discovered that let-7a was the first known human miRNA. Let-7a is originated from the nematode Caenorhabditis elegans and controls the time of stem cell division and differentiation [6,7]. Let-7a and its family members are highly maintained for species in sequences and functions, while error adjustment of let-7a can lead to a lower degree of cell differentiation and the development of cellular diseases such as cancers [8,9]. Due to the clinical significance of let-7a, many methods for detecting let-7a have been developed. Traditional methods for miRNA detections mainly include reverse transcription quantitative PCR (RT-qPCR) [10], electrophoresis [11], and microarrays [12]. However, these methods have the disadvantages of complex operational steps and requirement of sophisticated instrumentation and multiple separations. Some alternative miRNA
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sensing methods, which include fluorescent [13,14], colorimetric [15] and electrochemical [16–18] approaches, have recently been developed. Among them, electrochemical sensors have received more and more attentions due to their simple, cheap equipment, high sensitivity and efficiency response. In addition, different signal amplification technologies including rolling circle amplification (RCA) [19,20], hybridization chain reaction (HCR) [21,22], and catalytic hairpin assembly (CHA) [23–25] have been combined with these assay approaches to further improve the detection sensitivity. However, there are still some disadvantages, such as nuclease sensitivity to the sensing environment, requirement of optimal temperature (37 °C or higher) for enzymatic probe digestion, and increased assay cost with the use of enzymes [26]. Consequently, developing highly sensitive and convenient electrochemical sensors for miRNA detection is necessary. Self-assembly is a phenomenon, in which components (such as molecules) of a system are self-assembled and organized into a regular structure without the intervention of external forces [27]. The occurrence of a self-assembly process typically transforms the system from a disordered state to an ordered one, which can occur at different scales. Self-assembly is ubiquitous in nature. For example, cells of an organism
Corresponding author. E-mail address: [email protected] (Y. Xiang).
https://doi.org/10.1016/j.snb.2019.127068 Received 5 June 2019; Received in revised form 28 August 2019; Accepted 31 August 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Ronghui Ren, et al., Sensors & Actuators: B. Chemical, https://doi.org/10.1016/j.snb.2019.127068
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are self-assembled by various biomolecules. Self-assembly using various molecules is also an important method for constructing nanomaterials [28]. DNA molecular self-assembly based on the toehold strand displacement reaction (TSDR) is an important means to build nanostructures and nanodevices in dynamic DNA nanotechnology [29,30]. Yin et al. further extended the concept of triggering the self-assembly of several different metastable hairpin monomers to form a three-arm DNA junction by cascaded HCR using ssDNA sequences as the initiators [31]. This design requires only a small amount of DNA initiators to organize multiple sequences to form pre-designed DNA nanostructures, and the growth of the nanostructures highly depends on the amount of the added initiator. Therefore, this DNA-initiated self-assembled structure can provide a new possibility for achieving remarkably amplified signal. Herein, we report on the construction of an efficient sensing platform for the ultrasensitive and non-label electrochemical discrimination of miRNA. In such a miRNA sensing design, the target miRNA trigger sequences are recycled through a CHA process to form many trivalent DNAzyme junctions for efficient cleavage of the G-quadruplexlocked hairpins to produce a large amount of free G-quadruplex strands, which subsequently bind to hemin to produce remarkably enhanced current intensity because of electrochemical reduction characteristics of hemin for ultrasensitive monitoring of miRNAs in human serums in a completely label-free way.
the pretreated electrode and reacted at 25 °C for 2 h. After being thoroughly washed with immobilization buffer, MCH (1 mM) was dropped onto the SH-TP/AuE to incubate at 25 °C for 2 h for the formation of the SH-TP/MCH /AuE. 2.3. Amplified electrochemical assay of let-7a The HP1, HP2, HP3 were each diluted by 20 mM of Tris−HCl buffer (pH 7.4, 10 mM MgCl2, 10 mM KCl, 100 mM NaCl) to a final concentration of 5 μM and were separately processed by the same annealing process above. A series of let-7a at different concentrations were separately introduced into the mixture of HP1 (50 nM), HP2 (50 nM), and HP3 (50 nM) for 90 min at 25 °C. Subsequently, the above mixture was dropped onto the SH-TP/MCH/AuE and reacted at 25 °C for 30 min. Finally, hemin (0.2 mM) in 20 mM of HEPES buffer (1% DMSO, 200 mM NaCl, 50 mM KCl, pH 8.0) was reacted with the electrode for 30 min to induce the generation of the G-quadrulex/hemin complexes on the sensing surface after washing the electrode thoroughly with the buffer. The modified electrode was then ready for electrochemical measurement. 2.4. Electrochemical experimentations Cyclic voltammetry (CV) characterizations and differential pulse voltammetry (DPV) measurements were conducted on the CHI-621D electrochemical instrument, which included the platinum wire control electrode, the Ag/AgCl reference electrode, and the modified AuE working electrode. CV was carried out in 0.1 M KCl solution with the presence of 1 mM [Fe(CN)6]3−/4− from -0.1 V to 0.6 V at 50 mV s-1. Electrochemical impedance spectroscopy (EIS) was completed in 0.1 M KCl solution with the presence of 5 mM [Fe(CN)6]3−/4− in the frequency range of 0.1 to 105 Hz. DPV was completed in 20 mM of the HEPES buffer without DMSO by varying the potential from -0.15 V to -0.5 V. Prior to DPV, nitrogen was degassed into the HEPES buffer for 30 min to remove the dissolved O2.
2. Experimental 2.1. Chemicals and materials Hemin, 6-mercaptohexanol (MCH), dimethyl sulfoxide (DMSO), healthy human serum (Cat. No.: S1-M) and tris (2-carboxyethy) phosphine hydrochoride (TCEP) were ordered from Sigma-Aldrich (St. Louis, MO). Tris−HCl, HEPES and the oligonucleotides were purchased from Shanghai Sangon Biotechnology Inc. All reagents involved in this work were analytical grade. The ultrapure water used throughout the work was under 18.2 MΩ cm of resistance. DMSO-diluted hemin at the concentration of 10 mM was stored at −20 °C in the dark. The oligonucleotide sequences (from 5′ to 3′) were listed as follows. Let-7a: UGA GGU AGU AGG UUG UAU AGU U; Let-7b: UGA GGU AGU AGG UUG UGU GGU U; Let-7c: UGA GGU AGU AGG UUG UAU GGU U; Let-7e: UGA GGU AGG AGG UUG UAU AGU U; Hairpin probe 1 (HP1): CAT CTC TTC AGC GAT AGG TTG TAT AGT TAA GGT ATT AAA ACT ATA CA ACC TAC TAC CTC AAT CAC CCA TGT TAG TTA GT; Hairpin probe 2 (HP2): CAT CTC TTC AGC GAT GTT AAG GTA TTA ATT GAG GTA GTA CAT TAA TAC CTT AAC TAT ACA ACC TCA CCC ATG T; Hairpin probe 3 (HP3): CAT CTC TTC AGC GAT ATT GAG GTA GTA GGT TGT ATA GTT CCT ACT ACC TCA ATT AAT ACC TTA ACC ACC CAT GTT AGT TAG T; Thiolated probe (SH-TP): ACC CGC CCG TTT TAC TAA CTA TrAG GAA GAG ATG GGG TAG GGC GGG TTG GGT TTT-(CH2)6-SH.
2.5. Native polyacrylamide electrophoresis (PAGE) The reaction samples were loaded on an 8% native polyacrylamide gel for the electrophoresis experiment in 1 × TBE buffer. Electrophoresis was carried out under the voltage of 120 V for 40 min, after which the gel was stained by Gel-Red and imagined by the Bio-Rad imaging system (Hercules, CA, USA). 3. Results and discussion 3.1. The Principle of the proposed approach for miRNA detection Scheme 1 illustrates the mechanism for signal amplified electrochemical monitoring of let-7a based on target-triggered, cascaded and CHA for the formation of the Mg2+-dependent trivalent DNAzyme junctions. The system contains three DNA hairpins and a thiolated probe (SH-TP) that has a ribonucleobase (rA) recognition site for the Mg2+-dependent DNAzymes. Each hairpin contains three fragments: the brownoverhang region fully complementary to the SH-TP, the Mg2+-dependent DNAzyme catalytic core (the pink region), and the fully complementary regions between the three hairpins (the red, purple and green sequences with identical colors). SH-TP with the 5′ terminus modified with the thiol moiety is assembled on the sensing surface via the Au-S interaction. The loop region of the SH-TP contains the recognition sequence for the Mg2+-dependent DNAzymes and the stem region has the G-quadruplex sequence at the 5′ terminus, which can form the G-quadrulex/hemin complex with K+. The G-quadruplex strand is partially locked in the stem region through base pairing in SHTP to restrain the generation of the G-quadruplex/hemin without the
2.2. Preparation of the electrochemical biosensor First, a 3 mm diameter goldelectrode (AuE) was soaked in a piranha solution (30% H2O2 and 98% H2SO4 at a volume ratio of 1:3) for 30 min. The AuE was cleaned thoroughly with ultrapure water and further polished with alumina slurry (0.3 μm and 0.05 μm, respectively), followed by removing the residues by continuous sonication in ultrapure water three times and further by electrochemical scanning in H2SO4 (0.5 M) from -0.3 to 1.55 V. Finally, the treated electrode was dried with a nitrogen stream. The SH-TP probes in 20 mM of Tris−HCl immobilization buffer (100 mM NaCl, pH 7.4) were heated to 95 °C for 10 min and then slowly cooled to 25 °C for 2 h to ensure the formation stable hairpin structures. This was followed by the introduction of 10 mM TCEP to incubate for 1 h. Subsequently 10 μL of the above complexes (0.3 μM) was added on 2
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Scheme 1. Target-triggered formation of trivalent DNAzyme junctions for signal amplified electrochemical monitoring of miRNAs.
target miRNA trigger sequences. Therefore, HP1, HP2 and HP3 cannot assemble to produce the Mg2+-dependent DNAzyme catalytic cores without the existence of the target let-7a, and the ribonucleobase (rA) recognition site in the SH-TP cannot be cleaved to release the Gquadruplex sequences. Hemin is incapable of associating with the Gquadruplex to generate the G-quadruplex/hemin to confine hemin within the vicinity of the electrode. Therefore, a minimized background current response is produced. However, when the let-7a is reacted with the HP1, HP2 and HP3, let-7a first hybridizes with the complementary sequence of H1 (the red sequence) and unfolds H1. The green sequence of opened H1 further binds to the green sequence of H2 and unfolds H2. Likewise, H3 can be opened, which again unfolds H1 to trigger the CHA process for the formation of the junction structure with three Mg2+dependent DNAzymes (the trivalent DNAzyme junction) at the termini of HP1, HP2, and HP3. Subsequently, the DNAzyme junctions hybridize with the SH-TP substrate hairpins immobilized on the electrode, and under the assistance of the Mg2+ ions, the SH-TP can be cleaved at the ribonucleobase (rA) recognition sites to release the G-quadruplex sequences. Moreover, the trivalent DNAzyme junctions with multiple Mg2+-dependent DNAzyme structures can therefore efficiently cleave SH-TP to liberate many G-quadruplex sequences, which can be bind to lots of hemin to produce substantial current signal for electrochemical discrimination of miRNAs with high sensitivity.
peak currents slightly increased (curve c vs. b) due to nonspecific leakage of self-assembly of the three hairpins without existence of the initiator miRNA sequences [31]. However, the reaction of the mixture of let-7a, HP1, HP2 and HP3 with SH-TP/MCH/AuE brings apparent recovery of the peak currents (curve d) because the SH-TP hairpins are cleaved to form single strand DNAs on electrode surface, which reduces the electrostatic repulsion effect between the [Fe(CN)6]3−/4− species and the DNA backbones and the steric hindrance as well. In addition, EIS has also been performed to verify the sensor construction process as well. As displayed in Fig. 1B, a very low charge transfer (Rct) resistance (curve a) is observed on the bare AuE because of the excellent conductivity of the bare AuE. The immobilization of SH-TP and MCH on the electrode leads to the introduction of negative charges by the phosphate backbones of SH-TP and therefore results in a significantly increased Rct (curve b). The incubation of the mixture of HP1, HP2 and HP3 with SHTP/MCH/AuE shows no obvious change in Rct (curve c) while clear decrease in Rct can be obtained with the addition of the target let-7a to the mixture (curve d), indicating the cleavage of the SH-TP on the sensor electrode by the DNAzymes. These findings are in well agreement with the CV characterizations, which implies that the sensor is successfully designed and can be applied to measure let-7a.
3.2. CV and EIS characterizations
For conforming the potentiality of the senor for signal enhanced and ultrasensitive electrochemical measurement of let-7a based on the trivalent DNAzyme junctions, the current intensities of different mixtures were recorded. As displayed in Fig. 2A, the solution containing H1, H2 and H3 without the presence of let-7a shows a small background current signal (curve a) when incubated for 50 min and followed by subsequent reaction with 0.2 mM of hemin, implying minor cross reaction between the hairpins at room temperature. The mixture of let-7a and H1 shows an insignificant change in current intensity (curve b vs. a) because let-7a and H1 cannot produce the Mg2+-dependent DNAzyme catalytic core. The mixture of let-7a, H1 and H2 (without the addition of H3) causes a significant change in current intensity (curve c vs. b) because let-7a is able to open H1, and then H1 can hybridize with H2 to form the Mg2+-dependent DNAzymes to cleave SH-TP to release the G-
3.3. Feasibility of the sensor for detecting miRNA
CV is an effective electrochemical characterization method and is commonly used to characterize electrode surface modification processes. As shown in Fig. 1A, we can observe on the bare AuEs two welldefined, reversible current peaks (curve a) because of efficient electron transfer from the [Fe(CN)6]3−/4− probes to bare AuEs. However, the current signal sharply declines after being immobilized with SH-TP hairpins and blocked with MCH (curve b vs. a). One reason is due to the negative charges on the phosphate skeletons to repel the [Fe(CN)6]3-/4species from the sensing surface. The other reason is the steric hindrance caused by SH-TP in the hairpin structure of that hinders the [Fe (CN)6]3-/4- probes from approaching the sensing surface. After incubating the mixture of HP1, HP2 and HP3 with SH-TP/MCH/AuE, the 3
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Fig. 1. (A) CV and (B) EIS curves of (a) bare AuE, (b) SH-TP/ MCH/AuE, (c) SH-TP/MCH/AuE reacted with (HP1+HP2+HP3) and (d) SH-TP/MCH/AuE reacted with (let-7a+HP1+HP2+HP3). Let-7a was first reacted with the mixture of HP1, HP2 and HP3 for 90 min, followed by further reaction with SH-TP/MCH/AuE for 50 min. The concentrations of SH-TP, let-7a and the hairpins were 0.2 μM, 1 nM and 50 nM, respectively.
quadruplex sequence for binding with hemin. Importantly, the reaction of the target let-7a with the solution containing H1, H2 and H3 results in a substantial increase in current signal (curve d vs. c). Such an increase is essentially because of the CHA formation of many trivalent Mg2+-dependent DNAzymes for effective cleavage of SH-TP and subsequent capture of hemin on the sensing electrode. The detection of the target let-7a by our designed mechanism was further verified by the native PAGE experiment. According to Fig. 2B, the mixture of HP1, HP2 and HP3 shows a clear mixed band (Lane 5) because of the similar lengths of the three hairpins (Lanes 1–3), and the its incubation with SH-TP causes no change in band mobility of SH-TP (Lane 6 vs. Lane 4), which means no obvious cross reactions among these hairpins. The incubation of let-7a with the mixture of HP1, HP2 and HP3 leads to the formation of a new band (Lane 7) with highly decreased electrophoretic mobility and the decrease in band intensity of the hairpins, suggesting the assembly of the three hairpins triggered by the let-7a. The disappearance of the band of SH-TP (Lane 8) upon its introduction with previous mixture indicates the successful cleavage of SH-TP by the selfassembled DNAzyme junctions. These results demonstrate the capability of the trivalent DNAzyme structures for ultrasensitive monitoring of let-7a.
concentration of ST-HP at 0.3 μM. According to the results displayed in Fig. 3B (curve a), the current signal increases along the DNAzyme cleavage time and tends to stabilize after 30 min (with the incubation of let-7a with the mixed solution containing H1, H2 and H3 for the formation of trivalent DNAzymes). For comparison, the incubation of let7a with H1 and H2 only results in the formation of a single DNAzyme and the saturation of the peak current after 60 min (curve b), indicating that the trivalent DNAzymes can result in efficient cleavage of SH-TP and reduce the assay time. Therefore, the optimal DNAzyme cleavage time for let-7a determination is 30 min. 3.5. Target miRNA detection Under optimized assay parameters, the voltmmetric current signal to various concentrations of let-7a is shown in Fig. 4A. Current signals gradually increase along the concentration of let-7a from 0 to 1 nM (from a to i), indicating that the higher the concentration of let-7a can cause the formation of more Mg2+-dependent DNAzymes to cleave SHTP. Fig. 4B shows that the current signal is linearly related to the logarithmic concentration of let-7a from 1 fM to 1 nM. A linear regression equation of i (μA) = 4.1988 + 0.2588 lgc with a R2 of 0.9956 is obtained and the detection limit corresponding to the 3σ rule is 0.46 fM, which shows a significant improvement compared to most reported electrochemical methods for miRNA detection using complex signal amplification strategies or detection schemes (Table 1). In addition, for six repeated measurements at 1 nM of let-7a, the relative standard deviation is 4.4%, indicating excellent reproducibility of the sensor.
3.4. Experimental condition optimizations In order to acquire the best experimental conditions, we investigated the concentration of ST-HP and the Mg2+-dependent DNAzyme cleavage time, respectively, based on the current signal of the sensors to let-7a (1 nM). The concentration of ST-HP was first investigated from 0.1 to 0.4 μM with fixed DNAzyme cleave time of 50 min. As displayed in Fig. 3A, as the concentration of ST-HP increases (from 0.1 to 0.3 μM), the peak current first increases and then decreases (after 0.3 μM). This is because the increasing concentration of SH-TP can lead to the production of more G-qradruplex sequences upon DNAzyme cleavage. However, a higher concentration of ST-HP (above 0.3 μM) can relatively increase steric hindrances for the binding between the Mg2+-dependent DNAzymes and ST-HP, and thus decreased current responses are observed. These results suggest an optimal
3.6. Selectivity investigations To assess the discrimination ability of such a sensor to other members of the let-7 miRNA family (let-7b, let-7c, let-7e), which differ by only one or two bases in sequence to let-7a, the selective experiments were performed. The results are shown in the Fig. 5, from which we can see that the DPV response of the let-7a target (1 nM) is much higher than those generated by 10 nM of other let-7 miRNA family Fig. 2. (A) DPV signals of the SH-TP/MCH/AuE sensor for (a) HP1+HP2+HP3, (b) let-7a+HP1, (c) let-7a+HP1+HP2, and (d) let-7a+HP1+HP2+HP3. Let-7a was first incubated with the hairpins for 90 min, and then the resulting mixture was reacted with the sensor for 50 min. This was followed the reaction in solution containing hemin for 30 min and DPV measurements. The concentrations of SH-TP, let-7a, hemin and the hairpins were 0.2 μM, 1 nM, 0.2 mM and 50 nM, respectively. (B) Native PAGE of different reaction mixtures. Lane 1: HP1; Lane 2: HP2; Lane 3: HP3; Lane 4: SH-TP; Lane 5: HP1+HP2+HP3; Lane 6: HP1+HP2+HP3+SH-TP; Lane 7: let-7a+HP1+HP2+HP3; Lane 8: let-7a+HP1+HP2+HP3+SH-TP. The concentrations of let-7a and the hairpins were 1 nM and 200 nM, respectively.
4
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Fig. 3. Optimization of (A) the concentration of the SH-TP probe and (B) the DNAzyme cleavage time for (a) let-7a+HP1+HP2+HP3 and (b) let-7a+HP1+HP2 on the current signals of the sensing electrode. Error bars, SD, n = 3.
member sequences because of inhibition of the CHA process by the mismatched bases in the sequences. Moreover, the mixture of the target and base-mismatch sequences has a close peak current to the presence of the let-7a alone. These results clearly demonstrate that the Mg2+dependent DNAzyme assembly amplification method shows excellent performance to readily discriminate the miRNA family members with sequence homology.
Table 1 Different amplification strategies for electrochemical miRNA detection.
3.7. Detection of miRNA in human serums
Amplification Strategy
Linear range
Detection limit
References
Toehold-mediated strand displacement reactions Functionalized MOFs Polypyrrole nanowires Non-linear HCR CHA CHA
5 fM˜500 pM
1.4 fM
[32]
10 fM˜10 pM 100 fM˜1 nM 1 fM˜10 pM 500 fM˜12.5 nM 1 fM˜1 nM
3.6 fM 33 fM 0.33 fM 290 fM 0.46 fM
[33] [34] [35] [36] This work
In order to examine the effect of the constructed electrochemical miRNA sensor for monitoring the level of let-7a in serums, 10-fold diluted serums spiked with various concentrations of let-7a (10, 100 and 500 pM) were analyzed with the developed sensor. The results listed in Table 2 indicate that the recoveries and relative standard deviations for the added let-7a are between 97% and 103.4% and 3.3%–4.8%, respectively, indicating that the developed sensor has a potential application for diluted human serum samples. 4. Conclusion In summary, an electrochemical biosensor based on trivalent Mg2+dependent DNAzymes and let-7a triggered, cascaded and CHA of hairpin DNA for ultrasensitive detection of miRNA has been successfully constructed. Such a miRNA sensing approach shows the novelty of transforming the presence of the target molecules into the formation of new trivalent DNAzyme junctions via target-triggered assembly of three elaborately designed hairpins for highly efficient cleavage of the DNA substrates to reduce the assay time and to yield numerous signal sequences for drastic signal amplification. Moreover, simple, yet significant, signal enhancement without the involvement of nanomaterials/enzymes is realized by the integration of the target recycling (via target-triggered CHA) and metal ion-dependent DNAyzme amplification for achieving high sensitivity. The sensor can also be used to distinguish let-7a from sequence homology of miRNA family members.
Fig. 5. Specificity examination of the sensor for the target let-7a (1 nM) against other single/double-base mismatched sequences of let-7b, let-7c and let-7e at 10 nM each. Error bars, SD, n = 3. Table 2 Results for let-7a detection in diluted human serum samples (n = 6). Sample
Added (pM)
Founda (pM)
Recovery (%)
RSD (%)
1 2 3
10 100 500
9.7 103.4 513.5
97 103.4 102.7
3.3 4.1 4.8
Fig. 4. (A) DPV current signals of the sensors to various concentrations of let-7a. From a to i: 0, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 500 pM, 1 nM. (B) The calibration curve for peak current vs. lgc of let-7a from 1 fM to 1 nM. Error bars, SD, n = 3. 5
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Compared with other detection technologies, our sensing method has the advantages of high efficiency, simplicity, high sensitivity and easy operation, making the sensor a powerful and versatile platform. By carefully designing the recognition probes, the developed sensor can be extended to easily monitor different target molecules.
[19] [20]
Acknowledgments
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This work was supported by National Natural Science Foundation of China (No. 21675128) and Fundamental Research Funds for the Central Universities (XDJK2017A001).
[22]
References
[23]
[1] I.J. MacRae, K. Zhou, F. Li, A. Repic, A.N. Brooks, W.Z. Cande, P.D. Adams, J.A. Doudna, Structural basis for double-stranded RNA processing by dicer, Science 311 (2006) 195–198. [2] Y. Fan, X. Chen, A.D. Trigg, C. Tung, J. Kong, Z. Gao, Detection of microRNAs using target-guided formation of conducting polymer nanowires in nanogaps, J. Am. Chem. Soc. 129 (2007) 5437–5443. [3] N.J. Martinez, R.I. Gregory, MicroRNA gene regulatory pathways in the establishment and maintenance of ESC identity, Cell Stem Cell 7 (2010) 31–35. [4] Q. Sun, V. Tripathi, J.H. Yoon, D.K. Singh, Q. Hao, K.W. Min, S. Davila, R.W. Zealy, X. Li, M. Polycarpou-Schwarz, E. Lehrmann, Y. Zhang, K.G. Becker, S.M. Freier, Y. Zhu, S. Diederichs, S.G. Prasanth, A. Lal, M. Gorospe, K.V. Prasanth, MIR100 host gene-encoded lncRNAs regulate cell cycle by modulating the interaction between HuR and its target mRNAs, Nucleic Acids Res. 46 (2018) 10405–10416. [5] A.M. Krichevsky, G. Gabriely, miR-21: a small multi-faceted RNA, J. Cell. Mol. Med. 13 (2009) 39–53. [6] S.M. Lehmann, C. Krüger, B. Park, K. Derkow, K. Rosenberger, J. Baumgart, T. Trimbuch, G. Eom, M. Hinz, D. Kaul, P. Habbel, R. Kälin, E. Franzoni, A. Rybak, D. Nguyen, R. Veh, O. Ninnemann, O. Peters, R. Nitsch, F.L. Heppner, D. Golenbock, E. Schott, H.L. Ploegh, F.G. Wulczyn, S. Lehnardt, An unconventional role for miRNA: let-7 activates toll-like receptor 7 and causes neurodegeneration, Nat. Neurosci. 15 (2012) 827–837. [7] H. Lee, S. Han, C.S. Kwon, D. Lee, Biogenesis and regulation of thelet-7 miRNAs and their functional implications, Protein Cell 7 (2016) 100–113. [8] K. Liu, C. Zhang, T. Li, Y. Ding, T. Tu, F. Zhou, W. Qi, H. Chen, X. Sun, Let-7a inhibits growth and migration of breast cancer cells by targeting HMGA1, Int. J. Oncol. 46 (2015) 2526–2534. [9] B. Li, P. Chen, Y. Chang, J. Qi, H. Fu, H. Guo, Let-7a inhibits tumor cell growth and metastasis by directly targeting RTKN in human colon cancer, Biochem. Biophys. Res. Commun. 478 (2016) 739–745. [10] P. Mestdagh, P.V. Vlierberghe, A.D. Weer, D. Muth, F. Westermann, F. Speleman, J. Vandesompele, A novel and universal method for microRNA RT-qPCR data normalization, Genome Biol. 10 (2009) 64–74. [11] F. Yu, Q. Zhao, D. Zhang, Z. Yuan, H. Wang, Affinity interactions by capillary electrophoresis: binding, separation, and detection, Anal. Chem. 91 (2019) 372–387. [12] R. García-Álvarez, M. Hadjidemetriou, A. Sánchez-Iglesias, L.M. Liz-Marzán, K. Kostarelos, In vivoformation of protein corona on gold nanoparticles. The effect of their size and shape, Nanoscale 10 (2018) 1256–1264. [13] D. He, X. He, X. Yang, H. Li, A smart ZnO@polydopamine-nucleic acid nanosystem for ultrasensitive live cell mRNA imaging by the target-triggered intracellular selfassembly of active DNAzyme nanostructures, Chem. Sci. 8 (2017) 2832–2840. [14] Z. Ying, Z. Wu, B. Tu, W. Tan, J. Jiang, Genetically encoded fluorescent RNA sensor for ratiometric imaging of microRNA in living tumor cells, J. Am. Chem. Soc. 139 (2017) 9779–9782. [15] L. Zou, R. Li, M. Zhang, Y. Luo, N. Zhou, J. Wang, L. Ling, A colorimetric sensing platform based upon recognizing hybridization chain reaction products with oligonucleotide modified gold nanoparticles through triplex formation, Nanoscale 9 (2017) 1986–1992. [16] X. Li, B. Dou, R. Yuan, Y. Xiang, Mismatched catalytic hairpin assembly and ratiometric strategy for highly sensitive electrochemical detection of microRNA from tumor cells, Sens. Actuators B Chem. 286 (2019) 191–197. [17] J. Yang, Y. Wu, C. Gan, R. Yuan, Y. Xiang, Target-programmed and autonomous proximity binding aptasensor for amplified electronic detection of thrombin, Biosens. Bioelectron. 117 (2018) 743–747. [18] S. Xue, Q. Li, L. Wang, W. You, J. Zhang, R. Che, Copper-and Cobalt-Codoped CeO2
[24] [25] [26] [27] [28] [29] [30] [31] [32]
[33] [34] [35]
[36]
nanospheres with abundant oxygen vacancies as highly efficient electrocatalysts for dual-mode electrochemical sensing of microRNA, Anal. Chem. 91 (2019) 2659–2666. W. Zhou, D. Li, R. Yuan, Y. Xiang, Programmable DNA ring/hairpin-constrained structure enables ligation-free rolling circle amplification for imaging mRNAs in single cells, Anal. Chem. 91 (2019) 3628–3635. X. Liu, M. Zou, D. Li, R. Yuan, Y. Xiang, Hairpin/DNA ring ternary probes for highly sensitive detection and selective discrimination of microRNA among family members, Anal. Chim. Acta 1076 (2019) 138–143. K. Quan, J. Li, J. Wang, N. Xie, Q. Wei, J. Tang, X. Yang, K. Wang, J. Huang, DualmicroRNA-controlled double-amplified cascaded logic DNA circuits for accurate, discrimination of cell subtypes, Chem. Sci. 10 (2019) 1442–1449. X. Fan, Y. Qi, Z. Shi, Y. Lv, Y. Guo, A graphene-based biosensor for detecting microRNA with augmented sensitivity through helicase-assisted signal amplification of hybridization chain reaction, Sens. Actuators B Chem. 255 (2018) 1582–1586. A.P.K.K.K. Mudiyanselage, Q. Yu, M.A. Leon-Duque, B. Zhao, R. Wu, M. You, Genetically encoded catalytic hairpin assembly for sensitive RNA imaging in live cells, J. Am. Chem. Soc. 140 (2018) 8739–8874. Z. Wu, H. Fan, N.S.R. Satyavolu, W. Wang, R. Lake, J. Jiang, Y. Lu, Imaging endogenous metal ions in living cells using a DNAzyme–catalytic hairpin assembly Probe, Angew. Chem. 129 (2017) 8847–8851. S. Bi, S. Yue, Q. Wu, J. Ye, Triggered and catalyzed self-assembly of hyperbranched DNA structures for logic operations and homogeneous CRET biosensing of microRNA, Chem. Commun. (Camb.) 52 (2016) 5455–5458. J. Hu, Y. Li, Y. Li, B. Tang, C. Zhang, Single Quantum Dot-Based Nanosensor for Sensitive Detection of O-GlcNAc Transferase Activity, Anal. Chem. 89 (2017) 12992–12999. A. Lazcano, Prebiotic evolution and self-assembly of nucleic acids, ACS Nano 12 (2018) 9643–9647. H. He, J. Dai, Y. Meng, Z. Duan, C. Zhou, B. Zheng, J. Du, Y. Guo, D. Xiao, Selfassembly of DNA nanoparticles through multiple catalyzed hairpin assembly for enzyme-free nucleic acid amplified detection, Talanta 179 (2018) 641–645. S. Ou, T. Xu, X. Liu, X. Yu, R. Li, J. Deng, J. Yuan, Y. Chen, Rapid and ultrasensitive detection of microRNA based on strand displacement amplification-mediated entropy-driven circuit reaction, Sens. Actuators B Chem. 255 (2018) 3057–3063. W. Zhou, D. Li, Y. Chai, R. Yuan, Y. Xiang, RNA responsive and catalytic self-assembly of DNA nanostructures for highly sensitive fluorescence detection of microRNA from cancer cells, Chem. Commun. (Camb.) 51 (2015) 16494–16497. P. Yin, H.M.T. Choi, C.R. Calvert, N.A. Pierce, Programming biomolecular self-assembly pathways, Nature 451 (2018) 318–322. K. Shi, B. Dou, C. Yang, Y. Chai, R. Yuan, Y. Xiang, DNA-fueled molecular machine enables enzyme-free target recycling amplification for electronic detection of microRNA from cancer cells with highly minimized background noise, Anal. Chem. 87 (2015) 8578–8583. J. Chang, X. Wang, J. Wang, H. Li, F. Li, Nucleic acid-functionalized metal−organic framework-base homogeneous electrochemical biosensor for simultaneous detection of multiple tumor biomarkers, Anal. Chem. 91 (2019) 3604–3610. J. Wang, N. Hui, Electrochemical functionalization of polypyrrole nanowires for the development of ultrasensitive biosensors for detecting microRNA, Sens. Actuators B Chem. 281 (2019) 478–485. L. Zhou, Y. Wang, C. Yang, H. Xu, J. Luo, W. Zhang, X. Tang, S. Yang, W. Fu, K. Chang, M. Chen, A label-free electrochemical biosensor for microRNAs detection based on DNA nanomaterial by coupling with Y-shaped DNA structure and nonlinear hybridization chain reaction, Biosens. Bioelectron. 126 (2019) 657–663. Q. Li, F. Zeng, N. Lyu, J. Liang, Highly sensitive and specific electrochemical biosensor for microRNA-21 detection by coupling catalytic hairpin assembly with rolling circle amplification, Analyst 143 (2018) 2304–2309.
Ronghui Ren is a MS candidate in the School of Chemistry and Chemical Engineering at Southwest University in China. Qian Bi is a MS candidate in the School of Chemistry and Chemical Engineering at Southwest University in China. Ruo Yuan is a professor of chemistry at Southwest University, China. He received his PhD degree in analytical chemistry from Hunan University (China) in 1994. The main research interests of professor Yuan are chemical sensors and biosensors. Yun Xiang is a professor of chemistry at Southwest University in China. He received his PhD degree in chemistry from Arizona State University (USA) in 2008. His research focus is on biosensors
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
An efficient, label-free and sensitive electrochemical microRNA sensor based on target-initiated catalytic hairpin assembly of trivalent DNAzyme junctions Ronghui Ren, Qian Bi, Ruo Yuan, Yun Xiang
⁎
Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
ARTICLE INFO
ABSTRACT
Keywords: Catalytic hairpin assembly Electrochemistry DNAzyme Biosensor MicroRNA
We have reported an efficient and ultrasensitive electrochemical sensor for label-free sensing of microRNAs (miRNAs) in serum samples via the formation of trivalent DNAzyme junctions through a target-initiated catalytic hairpin assembly (CHA) approach. The target miRNA sequences can lead to CHA of three hairpins into many trivalent DNAzyme junctions, which exhibit significantly enhanced efficiency over the conventional single DNAzyme structure for cleaving the G-quadruplex-containing DNA substrate sequences on the sensor surface. Such a cyclic and efficient cleavage thus results in the generation of massive free G-quadruplex sequences, which subsequently bind and confine the electroactive species, hemin, in the vicinity of the electrode for yielding drastically enhanced current intensity for ultrasensitive sensing of the target from 1 fM to 1 nM with a low detection limit of 0.46 fM. Besides, the sensor can discriminate single- and two-base mismatched sequences among the let-7 family with excellent selectivity and can be used to monitor the target let-7a in diluted serum sample, making such a sensor hold great potentials for highly sensitive and facile discrimination of other miRNA biomarkers.
1. Introduction MicroRNAs (miRNAs) with about 17 to 25 nucleotides in length are a kind of endogenous regulatory non-coding RNAs found in eukaryotes [1,2]. Evidences have shown that specifically expressed miRNAs are associated with many significant diseases and these specific miRNAs are used as potential biomarkers to diagnose these diseases [3–5]. It was subsequently discovered that let-7a was the first known human miRNA. Let-7a is originated from the nematode Caenorhabditis elegans and controls the time of stem cell division and differentiation [6,7]. Let-7a and its family members are highly maintained for species in sequences and functions, while error adjustment of let-7a can lead to a lower degree of cell differentiation and the development of cellular diseases such as cancers [8,9]. Due to the clinical significance of let-7a, many methods for detecting let-7a have been developed. Traditional methods for miRNA detections mainly include reverse transcription quantitative PCR (RT-qPCR) [10], electrophoresis [11], and microarrays [12]. However, these methods have the disadvantages of complex operational steps and requirement of sophisticated instrumentation and multiple separations. Some alternative miRNA
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sensing methods, which include fluorescent [13,14], colorimetric [15] and electrochemical [16–18] approaches, have recently been developed. Among them, electrochemical sensors have received more and more attentions due to their simple, cheap equipment, high sensitivity and efficiency response. In addition, different signal amplification technologies including rolling circle amplification (RCA) [19,20], hybridization chain reaction (HCR) [21,22], and catalytic hairpin assembly (CHA) [23–25] have been combined with these assay approaches to further improve the detection sensitivity. However, there are still some disadvantages, such as nuclease sensitivity to the sensing environment, requirement of optimal temperature (37 °C or higher) for enzymatic probe digestion, and increased assay cost with the use of enzymes [26]. Consequently, developing highly sensitive and convenient electrochemical sensors for miRNA detection is necessary. Self-assembly is a phenomenon, in which components (such as molecules) of a system are self-assembled and organized into a regular structure without the intervention of external forces [27]. The occurrence of a self-assembly process typically transforms the system from a disordered state to an ordered one, which can occur at different scales. Self-assembly is ubiquitous in nature. For example, cells of an organism
Corresponding author. E-mail address: [email protected] (Y. Xiang).
https://doi.org/10.1016/j.snb.2019.127068 Received 5 June 2019; Received in revised form 28 August 2019; Accepted 31 August 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Ronghui Ren, et al., Sensors & Actuators: B. Chemical, https://doi.org/10.1016/j.snb.2019.127068
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are self-assembled by various biomolecules. Self-assembly using various molecules is also an important method for constructing nanomaterials [28]. DNA molecular self-assembly based on the toehold strand displacement reaction (TSDR) is an important means to build nanostructures and nanodevices in dynamic DNA nanotechnology [29,30]. Yin et al. further extended the concept of triggering the self-assembly of several different metastable hairpin monomers to form a three-arm DNA junction by cascaded HCR using ssDNA sequences as the initiators [31]. This design requires only a small amount of DNA initiators to organize multiple sequences to form pre-designed DNA nanostructures, and the growth of the nanostructures highly depends on the amount of the added initiator. Therefore, this DNA-initiated self-assembled structure can provide a new possibility for achieving remarkably amplified signal. Herein, we report on the construction of an efficient sensing platform for the ultrasensitive and non-label electrochemical discrimination of miRNA. In such a miRNA sensing design, the target miRNA trigger sequences are recycled through a CHA process to form many trivalent DNAzyme junctions for efficient cleavage of the G-quadruplexlocked hairpins to produce a large amount of free G-quadruplex strands, which subsequently bind to hemin to produce remarkably enhanced current intensity because of electrochemical reduction characteristics of hemin for ultrasensitive monitoring of miRNAs in human serums in a completely label-free way.
the pretreated electrode and reacted at 25 °C for 2 h. After being thoroughly washed with immobilization buffer, MCH (1 mM) was dropped onto the SH-TP/AuE to incubate at 25 °C for 2 h for the formation of the SH-TP/MCH /AuE. 2.3. Amplified electrochemical assay of let-7a The HP1, HP2, HP3 were each diluted by 20 mM of Tris−HCl buffer (pH 7.4, 10 mM MgCl2, 10 mM KCl, 100 mM NaCl) to a final concentration of 5 μM and were separately processed by the same annealing process above. A series of let-7a at different concentrations were separately introduced into the mixture of HP1 (50 nM), HP2 (50 nM), and HP3 (50 nM) for 90 min at 25 °C. Subsequently, the above mixture was dropped onto the SH-TP/MCH/AuE and reacted at 25 °C for 30 min. Finally, hemin (0.2 mM) in 20 mM of HEPES buffer (1% DMSO, 200 mM NaCl, 50 mM KCl, pH 8.0) was reacted with the electrode for 30 min to induce the generation of the G-quadrulex/hemin complexes on the sensing surface after washing the electrode thoroughly with the buffer. The modified electrode was then ready for electrochemical measurement. 2.4. Electrochemical experimentations Cyclic voltammetry (CV) characterizations and differential pulse voltammetry (DPV) measurements were conducted on the CHI-621D electrochemical instrument, which included the platinum wire control electrode, the Ag/AgCl reference electrode, and the modified AuE working electrode. CV was carried out in 0.1 M KCl solution with the presence of 1 mM [Fe(CN)6]3−/4− from -0.1 V to 0.6 V at 50 mV s-1. Electrochemical impedance spectroscopy (EIS) was completed in 0.1 M KCl solution with the presence of 5 mM [Fe(CN)6]3−/4− in the frequency range of 0.1 to 105 Hz. DPV was completed in 20 mM of the HEPES buffer without DMSO by varying the potential from -0.15 V to -0.5 V. Prior to DPV, nitrogen was degassed into the HEPES buffer for 30 min to remove the dissolved O2.
2. Experimental 2.1. Chemicals and materials Hemin, 6-mercaptohexanol (MCH), dimethyl sulfoxide (DMSO), healthy human serum (Cat. No.: S1-M) and tris (2-carboxyethy) phosphine hydrochoride (TCEP) were ordered from Sigma-Aldrich (St. Louis, MO). Tris−HCl, HEPES and the oligonucleotides were purchased from Shanghai Sangon Biotechnology Inc. All reagents involved in this work were analytical grade. The ultrapure water used throughout the work was under 18.2 MΩ cm of resistance. DMSO-diluted hemin at the concentration of 10 mM was stored at −20 °C in the dark. The oligonucleotide sequences (from 5′ to 3′) were listed as follows. Let-7a: UGA GGU AGU AGG UUG UAU AGU U; Let-7b: UGA GGU AGU AGG UUG UGU GGU U; Let-7c: UGA GGU AGU AGG UUG UAU GGU U; Let-7e: UGA GGU AGG AGG UUG UAU AGU U; Hairpin probe 1 (HP1): CAT CTC TTC AGC GAT AGG TTG TAT AGT TAA GGT ATT AAA ACT ATA CA ACC TAC TAC CTC AAT CAC CCA TGT TAG TTA GT; Hairpin probe 2 (HP2): CAT CTC TTC AGC GAT GTT AAG GTA TTA ATT GAG GTA GTA CAT TAA TAC CTT AAC TAT ACA ACC TCA CCC ATG T; Hairpin probe 3 (HP3): CAT CTC TTC AGC GAT ATT GAG GTA GTA GGT TGT ATA GTT CCT ACT ACC TCA ATT AAT ACC TTA ACC ACC CAT GTT AGT TAG T; Thiolated probe (SH-TP): ACC CGC CCG TTT TAC TAA CTA TrAG GAA GAG ATG GGG TAG GGC GGG TTG GGT TTT-(CH2)6-SH.
2.5. Native polyacrylamide electrophoresis (PAGE) The reaction samples were loaded on an 8% native polyacrylamide gel for the electrophoresis experiment in 1 × TBE buffer. Electrophoresis was carried out under the voltage of 120 V for 40 min, after which the gel was stained by Gel-Red and imagined by the Bio-Rad imaging system (Hercules, CA, USA). 3. Results and discussion 3.1. The Principle of the proposed approach for miRNA detection Scheme 1 illustrates the mechanism for signal amplified electrochemical monitoring of let-7a based on target-triggered, cascaded and CHA for the formation of the Mg2+-dependent trivalent DNAzyme junctions. The system contains three DNA hairpins and a thiolated probe (SH-TP) that has a ribonucleobase (rA) recognition site for the Mg2+-dependent DNAzymes. Each hairpin contains three fragments: the brownoverhang region fully complementary to the SH-TP, the Mg2+-dependent DNAzyme catalytic core (the pink region), and the fully complementary regions between the three hairpins (the red, purple and green sequences with identical colors). SH-TP with the 5′ terminus modified with the thiol moiety is assembled on the sensing surface via the Au-S interaction. The loop region of the SH-TP contains the recognition sequence for the Mg2+-dependent DNAzymes and the stem region has the G-quadruplex sequence at the 5′ terminus, which can form the G-quadrulex/hemin complex with K+. The G-quadruplex strand is partially locked in the stem region through base pairing in SHTP to restrain the generation of the G-quadruplex/hemin without the
2.2. Preparation of the electrochemical biosensor First, a 3 mm diameter goldelectrode (AuE) was soaked in a piranha solution (30% H2O2 and 98% H2SO4 at a volume ratio of 1:3) for 30 min. The AuE was cleaned thoroughly with ultrapure water and further polished with alumina slurry (0.3 μm and 0.05 μm, respectively), followed by removing the residues by continuous sonication in ultrapure water three times and further by electrochemical scanning in H2SO4 (0.5 M) from -0.3 to 1.55 V. Finally, the treated electrode was dried with a nitrogen stream. The SH-TP probes in 20 mM of Tris−HCl immobilization buffer (100 mM NaCl, pH 7.4) were heated to 95 °C for 10 min and then slowly cooled to 25 °C for 2 h to ensure the formation stable hairpin structures. This was followed by the introduction of 10 mM TCEP to incubate for 1 h. Subsequently 10 μL of the above complexes (0.3 μM) was added on 2
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Scheme 1. Target-triggered formation of trivalent DNAzyme junctions for signal amplified electrochemical monitoring of miRNAs.
target miRNA trigger sequences. Therefore, HP1, HP2 and HP3 cannot assemble to produce the Mg2+-dependent DNAzyme catalytic cores without the existence of the target let-7a, and the ribonucleobase (rA) recognition site in the SH-TP cannot be cleaved to release the Gquadruplex sequences. Hemin is incapable of associating with the Gquadruplex to generate the G-quadruplex/hemin to confine hemin within the vicinity of the electrode. Therefore, a minimized background current response is produced. However, when the let-7a is reacted with the HP1, HP2 and HP3, let-7a first hybridizes with the complementary sequence of H1 (the red sequence) and unfolds H1. The green sequence of opened H1 further binds to the green sequence of H2 and unfolds H2. Likewise, H3 can be opened, which again unfolds H1 to trigger the CHA process for the formation of the junction structure with three Mg2+dependent DNAzymes (the trivalent DNAzyme junction) at the termini of HP1, HP2, and HP3. Subsequently, the DNAzyme junctions hybridize with the SH-TP substrate hairpins immobilized on the electrode, and under the assistance of the Mg2+ ions, the SH-TP can be cleaved at the ribonucleobase (rA) recognition sites to release the G-quadruplex sequences. Moreover, the trivalent DNAzyme junctions with multiple Mg2+-dependent DNAzyme structures can therefore efficiently cleave SH-TP to liberate many G-quadruplex sequences, which can be bind to lots of hemin to produce substantial current signal for electrochemical discrimination of miRNAs with high sensitivity.
peak currents slightly increased (curve c vs. b) due to nonspecific leakage of self-assembly of the three hairpins without existence of the initiator miRNA sequences [31]. However, the reaction of the mixture of let-7a, HP1, HP2 and HP3 with SH-TP/MCH/AuE brings apparent recovery of the peak currents (curve d) because the SH-TP hairpins are cleaved to form single strand DNAs on electrode surface, which reduces the electrostatic repulsion effect between the [Fe(CN)6]3−/4− species and the DNA backbones and the steric hindrance as well. In addition, EIS has also been performed to verify the sensor construction process as well. As displayed in Fig. 1B, a very low charge transfer (Rct) resistance (curve a) is observed on the bare AuE because of the excellent conductivity of the bare AuE. The immobilization of SH-TP and MCH on the electrode leads to the introduction of negative charges by the phosphate backbones of SH-TP and therefore results in a significantly increased Rct (curve b). The incubation of the mixture of HP1, HP2 and HP3 with SHTP/MCH/AuE shows no obvious change in Rct (curve c) while clear decrease in Rct can be obtained with the addition of the target let-7a to the mixture (curve d), indicating the cleavage of the SH-TP on the sensor electrode by the DNAzymes. These findings are in well agreement with the CV characterizations, which implies that the sensor is successfully designed and can be applied to measure let-7a.
3.2. CV and EIS characterizations
For conforming the potentiality of the senor for signal enhanced and ultrasensitive electrochemical measurement of let-7a based on the trivalent DNAzyme junctions, the current intensities of different mixtures were recorded. As displayed in Fig. 2A, the solution containing H1, H2 and H3 without the presence of let-7a shows a small background current signal (curve a) when incubated for 50 min and followed by subsequent reaction with 0.2 mM of hemin, implying minor cross reaction between the hairpins at room temperature. The mixture of let-7a and H1 shows an insignificant change in current intensity (curve b vs. a) because let-7a and H1 cannot produce the Mg2+-dependent DNAzyme catalytic core. The mixture of let-7a, H1 and H2 (without the addition of H3) causes a significant change in current intensity (curve c vs. b) because let-7a is able to open H1, and then H1 can hybridize with H2 to form the Mg2+-dependent DNAzymes to cleave SH-TP to release the G-
3.3. Feasibility of the sensor for detecting miRNA
CV is an effective electrochemical characterization method and is commonly used to characterize electrode surface modification processes. As shown in Fig. 1A, we can observe on the bare AuEs two welldefined, reversible current peaks (curve a) because of efficient electron transfer from the [Fe(CN)6]3−/4− probes to bare AuEs. However, the current signal sharply declines after being immobilized with SH-TP hairpins and blocked with MCH (curve b vs. a). One reason is due to the negative charges on the phosphate skeletons to repel the [Fe(CN)6]3-/4species from the sensing surface. The other reason is the steric hindrance caused by SH-TP in the hairpin structure of that hinders the [Fe (CN)6]3-/4- probes from approaching the sensing surface. After incubating the mixture of HP1, HP2 and HP3 with SH-TP/MCH/AuE, the 3
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Fig. 1. (A) CV and (B) EIS curves of (a) bare AuE, (b) SH-TP/ MCH/AuE, (c) SH-TP/MCH/AuE reacted with (HP1+HP2+HP3) and (d) SH-TP/MCH/AuE reacted with (let-7a+HP1+HP2+HP3). Let-7a was first reacted with the mixture of HP1, HP2 and HP3 for 90 min, followed by further reaction with SH-TP/MCH/AuE for 50 min. The concentrations of SH-TP, let-7a and the hairpins were 0.2 μM, 1 nM and 50 nM, respectively.
quadruplex sequence for binding with hemin. Importantly, the reaction of the target let-7a with the solution containing H1, H2 and H3 results in a substantial increase in current signal (curve d vs. c). Such an increase is essentially because of the CHA formation of many trivalent Mg2+-dependent DNAzymes for effective cleavage of SH-TP and subsequent capture of hemin on the sensing electrode. The detection of the target let-7a by our designed mechanism was further verified by the native PAGE experiment. According to Fig. 2B, the mixture of HP1, HP2 and HP3 shows a clear mixed band (Lane 5) because of the similar lengths of the three hairpins (Lanes 1–3), and the its incubation with SH-TP causes no change in band mobility of SH-TP (Lane 6 vs. Lane 4), which means no obvious cross reactions among these hairpins. The incubation of let-7a with the mixture of HP1, HP2 and HP3 leads to the formation of a new band (Lane 7) with highly decreased electrophoretic mobility and the decrease in band intensity of the hairpins, suggesting the assembly of the three hairpins triggered by the let-7a. The disappearance of the band of SH-TP (Lane 8) upon its introduction with previous mixture indicates the successful cleavage of SH-TP by the selfassembled DNAzyme junctions. These results demonstrate the capability of the trivalent DNAzyme structures for ultrasensitive monitoring of let-7a.
concentration of ST-HP at 0.3 μM. According to the results displayed in Fig. 3B (curve a), the current signal increases along the DNAzyme cleavage time and tends to stabilize after 30 min (with the incubation of let-7a with the mixed solution containing H1, H2 and H3 for the formation of trivalent DNAzymes). For comparison, the incubation of let7a with H1 and H2 only results in the formation of a single DNAzyme and the saturation of the peak current after 60 min (curve b), indicating that the trivalent DNAzymes can result in efficient cleavage of SH-TP and reduce the assay time. Therefore, the optimal DNAzyme cleavage time for let-7a determination is 30 min. 3.5. Target miRNA detection Under optimized assay parameters, the voltmmetric current signal to various concentrations of let-7a is shown in Fig. 4A. Current signals gradually increase along the concentration of let-7a from 0 to 1 nM (from a to i), indicating that the higher the concentration of let-7a can cause the formation of more Mg2+-dependent DNAzymes to cleave SHTP. Fig. 4B shows that the current signal is linearly related to the logarithmic concentration of let-7a from 1 fM to 1 nM. A linear regression equation of i (μA) = 4.1988 + 0.2588 lgc with a R2 of 0.9956 is obtained and the detection limit corresponding to the 3σ rule is 0.46 fM, which shows a significant improvement compared to most reported electrochemical methods for miRNA detection using complex signal amplification strategies or detection schemes (Table 1). In addition, for six repeated measurements at 1 nM of let-7a, the relative standard deviation is 4.4%, indicating excellent reproducibility of the sensor.
3.4. Experimental condition optimizations In order to acquire the best experimental conditions, we investigated the concentration of ST-HP and the Mg2+-dependent DNAzyme cleavage time, respectively, based on the current signal of the sensors to let-7a (1 nM). The concentration of ST-HP was first investigated from 0.1 to 0.4 μM with fixed DNAzyme cleave time of 50 min. As displayed in Fig. 3A, as the concentration of ST-HP increases (from 0.1 to 0.3 μM), the peak current first increases and then decreases (after 0.3 μM). This is because the increasing concentration of SH-TP can lead to the production of more G-qradruplex sequences upon DNAzyme cleavage. However, a higher concentration of ST-HP (above 0.3 μM) can relatively increase steric hindrances for the binding between the Mg2+-dependent DNAzymes and ST-HP, and thus decreased current responses are observed. These results suggest an optimal
3.6. Selectivity investigations To assess the discrimination ability of such a sensor to other members of the let-7 miRNA family (let-7b, let-7c, let-7e), which differ by only one or two bases in sequence to let-7a, the selective experiments were performed. The results are shown in the Fig. 5, from which we can see that the DPV response of the let-7a target (1 nM) is much higher than those generated by 10 nM of other let-7 miRNA family Fig. 2. (A) DPV signals of the SH-TP/MCH/AuE sensor for (a) HP1+HP2+HP3, (b) let-7a+HP1, (c) let-7a+HP1+HP2, and (d) let-7a+HP1+HP2+HP3. Let-7a was first incubated with the hairpins for 90 min, and then the resulting mixture was reacted with the sensor for 50 min. This was followed the reaction in solution containing hemin for 30 min and DPV measurements. The concentrations of SH-TP, let-7a, hemin and the hairpins were 0.2 μM, 1 nM, 0.2 mM and 50 nM, respectively. (B) Native PAGE of different reaction mixtures. Lane 1: HP1; Lane 2: HP2; Lane 3: HP3; Lane 4: SH-TP; Lane 5: HP1+HP2+HP3; Lane 6: HP1+HP2+HP3+SH-TP; Lane 7: let-7a+HP1+HP2+HP3; Lane 8: let-7a+HP1+HP2+HP3+SH-TP. The concentrations of let-7a and the hairpins were 1 nM and 200 nM, respectively.
4
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Fig. 3. Optimization of (A) the concentration of the SH-TP probe and (B) the DNAzyme cleavage time for (a) let-7a+HP1+HP2+HP3 and (b) let-7a+HP1+HP2 on the current signals of the sensing electrode. Error bars, SD, n = 3.
member sequences because of inhibition of the CHA process by the mismatched bases in the sequences. Moreover, the mixture of the target and base-mismatch sequences has a close peak current to the presence of the let-7a alone. These results clearly demonstrate that the Mg2+dependent DNAzyme assembly amplification method shows excellent performance to readily discriminate the miRNA family members with sequence homology.
Table 1 Different amplification strategies for electrochemical miRNA detection.
3.7. Detection of miRNA in human serums
Amplification Strategy
Linear range
Detection limit
References
Toehold-mediated strand displacement reactions Functionalized MOFs Polypyrrole nanowires Non-linear HCR CHA CHA
5 fM˜500 pM
1.4 fM
[32]
10 fM˜10 pM 100 fM˜1 nM 1 fM˜10 pM 500 fM˜12.5 nM 1 fM˜1 nM
3.6 fM 33 fM 0.33 fM 290 fM 0.46 fM
[33] [34] [35] [36] This work
In order to examine the effect of the constructed electrochemical miRNA sensor for monitoring the level of let-7a in serums, 10-fold diluted serums spiked with various concentrations of let-7a (10, 100 and 500 pM) were analyzed with the developed sensor. The results listed in Table 2 indicate that the recoveries and relative standard deviations for the added let-7a are between 97% and 103.4% and 3.3%–4.8%, respectively, indicating that the developed sensor has a potential application for diluted human serum samples. 4. Conclusion In summary, an electrochemical biosensor based on trivalent Mg2+dependent DNAzymes and let-7a triggered, cascaded and CHA of hairpin DNA for ultrasensitive detection of miRNA has been successfully constructed. Such a miRNA sensing approach shows the novelty of transforming the presence of the target molecules into the formation of new trivalent DNAzyme junctions via target-triggered assembly of three elaborately designed hairpins for highly efficient cleavage of the DNA substrates to reduce the assay time and to yield numerous signal sequences for drastic signal amplification. Moreover, simple, yet significant, signal enhancement without the involvement of nanomaterials/enzymes is realized by the integration of the target recycling (via target-triggered CHA) and metal ion-dependent DNAyzme amplification for achieving high sensitivity. The sensor can also be used to distinguish let-7a from sequence homology of miRNA family members.
Fig. 5. Specificity examination of the sensor for the target let-7a (1 nM) against other single/double-base mismatched sequences of let-7b, let-7c and let-7e at 10 nM each. Error bars, SD, n = 3. Table 2 Results for let-7a detection in diluted human serum samples (n = 6). Sample
Added (pM)
Founda (pM)
Recovery (%)
RSD (%)
1 2 3
10 100 500
9.7 103.4 513.5
97 103.4 102.7
3.3 4.1 4.8
Fig. 4. (A) DPV current signals of the sensors to various concentrations of let-7a. From a to i: 0, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 500 pM, 1 nM. (B) The calibration curve for peak current vs. lgc of let-7a from 1 fM to 1 nM. Error bars, SD, n = 3. 5
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Compared with other detection technologies, our sensing method has the advantages of high efficiency, simplicity, high sensitivity and easy operation, making the sensor a powerful and versatile platform. By carefully designing the recognition probes, the developed sensor can be extended to easily monitor different target molecules.
[19] [20]
Acknowledgments
[21]
This work was supported by National Natural Science Foundation of China (No. 21675128) and Fundamental Research Funds for the Central Universities (XDJK2017A001).
[22]
References
[23]
[1] I.J. MacRae, K. Zhou, F. Li, A. Repic, A.N. Brooks, W.Z. Cande, P.D. Adams, J.A. Doudna, Structural basis for double-stranded RNA processing by dicer, Science 311 (2006) 195–198. [2] Y. Fan, X. Chen, A.D. Trigg, C. Tung, J. Kong, Z. Gao, Detection of microRNAs using target-guided formation of conducting polymer nanowires in nanogaps, J. Am. Chem. Soc. 129 (2007) 5437–5443. [3] N.J. Martinez, R.I. Gregory, MicroRNA gene regulatory pathways in the establishment and maintenance of ESC identity, Cell Stem Cell 7 (2010) 31–35. [4] Q. Sun, V. Tripathi, J.H. Yoon, D.K. Singh, Q. Hao, K.W. Min, S. Davila, R.W. Zealy, X. Li, M. Polycarpou-Schwarz, E. Lehrmann, Y. Zhang, K.G. Becker, S.M. Freier, Y. Zhu, S. Diederichs, S.G. Prasanth, A. Lal, M. Gorospe, K.V. Prasanth, MIR100 host gene-encoded lncRNAs regulate cell cycle by modulating the interaction between HuR and its target mRNAs, Nucleic Acids Res. 46 (2018) 10405–10416. [5] A.M. Krichevsky, G. Gabriely, miR-21: a small multi-faceted RNA, J. Cell. Mol. Med. 13 (2009) 39–53. [6] S.M. Lehmann, C. Krüger, B. Park, K. Derkow, K. Rosenberger, J. Baumgart, T. Trimbuch, G. Eom, M. Hinz, D. Kaul, P. Habbel, R. Kälin, E. Franzoni, A. Rybak, D. Nguyen, R. Veh, O. Ninnemann, O. Peters, R. Nitsch, F.L. Heppner, D. Golenbock, E. Schott, H.L. Ploegh, F.G. Wulczyn, S. Lehnardt, An unconventional role for miRNA: let-7 activates toll-like receptor 7 and causes neurodegeneration, Nat. Neurosci. 15 (2012) 827–837. [7] H. Lee, S. Han, C.S. Kwon, D. Lee, Biogenesis and regulation of thelet-7 miRNAs and their functional implications, Protein Cell 7 (2016) 100–113. [8] K. Liu, C. Zhang, T. Li, Y. Ding, T. Tu, F. Zhou, W. Qi, H. Chen, X. Sun, Let-7a inhibits growth and migration of breast cancer cells by targeting HMGA1, Int. J. Oncol. 46 (2015) 2526–2534. [9] B. Li, P. Chen, Y. Chang, J. Qi, H. Fu, H. Guo, Let-7a inhibits tumor cell growth and metastasis by directly targeting RTKN in human colon cancer, Biochem. Biophys. Res. Commun. 478 (2016) 739–745. [10] P. Mestdagh, P.V. Vlierberghe, A.D. Weer, D. Muth, F. Westermann, F. Speleman, J. Vandesompele, A novel and universal method for microRNA RT-qPCR data normalization, Genome Biol. 10 (2009) 64–74. [11] F. Yu, Q. Zhao, D. Zhang, Z. Yuan, H. Wang, Affinity interactions by capillary electrophoresis: binding, separation, and detection, Anal. Chem. 91 (2019) 372–387. [12] R. García-Álvarez, M. Hadjidemetriou, A. Sánchez-Iglesias, L.M. Liz-Marzán, K. Kostarelos, In vivoformation of protein corona on gold nanoparticles. The effect of their size and shape, Nanoscale 10 (2018) 1256–1264. [13] D. He, X. He, X. Yang, H. Li, A smart ZnO@polydopamine-nucleic acid nanosystem for ultrasensitive live cell mRNA imaging by the target-triggered intracellular selfassembly of active DNAzyme nanostructures, Chem. Sci. 8 (2017) 2832–2840. [14] Z. Ying, Z. Wu, B. Tu, W. Tan, J. Jiang, Genetically encoded fluorescent RNA sensor for ratiometric imaging of microRNA in living tumor cells, J. Am. Chem. Soc. 139 (2017) 9779–9782. [15] L. Zou, R. Li, M. Zhang, Y. Luo, N. Zhou, J. Wang, L. Ling, A colorimetric sensing platform based upon recognizing hybridization chain reaction products with oligonucleotide modified gold nanoparticles through triplex formation, Nanoscale 9 (2017) 1986–1992. [16] X. Li, B. Dou, R. Yuan, Y. Xiang, Mismatched catalytic hairpin assembly and ratiometric strategy for highly sensitive electrochemical detection of microRNA from tumor cells, Sens. Actuators B Chem. 286 (2019) 191–197. [17] J. Yang, Y. Wu, C. Gan, R. Yuan, Y. Xiang, Target-programmed and autonomous proximity binding aptasensor for amplified electronic detection of thrombin, Biosens. Bioelectron. 117 (2018) 743–747. [18] S. Xue, Q. Li, L. Wang, W. You, J. Zhang, R. Che, Copper-and Cobalt-Codoped CeO2
[24] [25] [26] [27] [28] [29] [30] [31] [32]
[33] [34] [35]
[36]
nanospheres with abundant oxygen vacancies as highly efficient electrocatalysts for dual-mode electrochemical sensing of microRNA, Anal. Chem. 91 (2019) 2659–2666. W. Zhou, D. Li, R. Yuan, Y. Xiang, Programmable DNA ring/hairpin-constrained structure enables ligation-free rolling circle amplification for imaging mRNAs in single cells, Anal. Chem. 91 (2019) 3628–3635. X. Liu, M. Zou, D. Li, R. Yuan, Y. Xiang, Hairpin/DNA ring ternary probes for highly sensitive detection and selective discrimination of microRNA among family members, Anal. Chim. Acta 1076 (2019) 138–143. K. Quan, J. Li, J. Wang, N. Xie, Q. Wei, J. Tang, X. Yang, K. Wang, J. Huang, DualmicroRNA-controlled double-amplified cascaded logic DNA circuits for accurate, discrimination of cell subtypes, Chem. Sci. 10 (2019) 1442–1449. X. Fan, Y. Qi, Z. Shi, Y. Lv, Y. Guo, A graphene-based biosensor for detecting microRNA with augmented sensitivity through helicase-assisted signal amplification of hybridization chain reaction, Sens. Actuators B Chem. 255 (2018) 1582–1586. A.P.K.K.K. Mudiyanselage, Q. Yu, M.A. Leon-Duque, B. Zhao, R. Wu, M. You, Genetically encoded catalytic hairpin assembly for sensitive RNA imaging in live cells, J. Am. Chem. Soc. 140 (2018) 8739–8874. Z. Wu, H. Fan, N.S.R. Satyavolu, W. Wang, R. Lake, J. Jiang, Y. Lu, Imaging endogenous metal ions in living cells using a DNAzyme–catalytic hairpin assembly Probe, Angew. Chem. 129 (2017) 8847–8851. S. Bi, S. Yue, Q. Wu, J. Ye, Triggered and catalyzed self-assembly of hyperbranched DNA structures for logic operations and homogeneous CRET biosensing of microRNA, Chem. Commun. (Camb.) 52 (2016) 5455–5458. J. Hu, Y. Li, Y. Li, B. Tang, C. Zhang, Single Quantum Dot-Based Nanosensor for Sensitive Detection of O-GlcNAc Transferase Activity, Anal. Chem. 89 (2017) 12992–12999. A. Lazcano, Prebiotic evolution and self-assembly of nucleic acids, ACS Nano 12 (2018) 9643–9647. H. He, J. Dai, Y. Meng, Z. Duan, C. Zhou, B. Zheng, J. Du, Y. Guo, D. Xiao, Selfassembly of DNA nanoparticles through multiple catalyzed hairpin assembly for enzyme-free nucleic acid amplified detection, Talanta 179 (2018) 641–645. S. Ou, T. Xu, X. Liu, X. Yu, R. Li, J. Deng, J. Yuan, Y. Chen, Rapid and ultrasensitive detection of microRNA based on strand displacement amplification-mediated entropy-driven circuit reaction, Sens. Actuators B Chem. 255 (2018) 3057–3063. W. Zhou, D. Li, Y. Chai, R. Yuan, Y. Xiang, RNA responsive and catalytic self-assembly of DNA nanostructures for highly sensitive fluorescence detection of microRNA from cancer cells, Chem. Commun. (Camb.) 51 (2015) 16494–16497. P. Yin, H.M.T. Choi, C.R. Calvert, N.A. Pierce, Programming biomolecular self-assembly pathways, Nature 451 (2018) 318–322. K. Shi, B. Dou, C. Yang, Y. Chai, R. Yuan, Y. Xiang, DNA-fueled molecular machine enables enzyme-free target recycling amplification for electronic detection of microRNA from cancer cells with highly minimized background noise, Anal. Chem. 87 (2015) 8578–8583. J. Chang, X. Wang, J. Wang, H. Li, F. Li, Nucleic acid-functionalized metal−organic framework-base homogeneous electrochemical biosensor for simultaneous detection of multiple tumor biomarkers, Anal. Chem. 91 (2019) 3604–3610. J. Wang, N. Hui, Electrochemical functionalization of polypyrrole nanowires for the development of ultrasensitive biosensors for detecting microRNA, Sens. Actuators B Chem. 281 (2019) 478–485. L. Zhou, Y. Wang, C. Yang, H. Xu, J. Luo, W. Zhang, X. Tang, S. Yang, W. Fu, K. Chang, M. Chen, A label-free electrochemical biosensor for microRNAs detection based on DNA nanomaterial by coupling with Y-shaped DNA structure and nonlinear hybridization chain reaction, Biosens. Bioelectron. 126 (2019) 657–663. Q. Li, F. Zeng, N. Lyu, J. Liang, Highly sensitive and specific electrochemical biosensor for microRNA-21 detection by coupling catalytic hairpin assembly with rolling circle amplification, Analyst 143 (2018) 2304–2309.
Ronghui Ren is a MS candidate in the School of Chemistry and Chemical Engineering at Southwest University in China. Qian Bi is a MS candidate in the School of Chemistry and Chemical Engineering at Southwest University in China. Ruo Yuan is a professor of chemistry at Southwest University, China. He received his PhD degree in analytical chemistry from Hunan University (China) in 1994. The main research interests of professor Yuan are chemical sensors and biosensors. Yun Xiang is a professor of chemistry at Southwest University in China. He received his PhD degree in chemistry from Arizona State University (USA) in 2008. His research focus is on biosensors
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