Versatile Electrochemiluminescence and Electrochemical âon-off

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Versatile Electrochemiluminescence and Electrochemical “on-off” Assays of Methyltransferases and Aflatoxin B1 Based on a Novel Multifunctional DNA Nanotube Junjun Ge, Yu Zhao, Chunli Li, and Guifen Jie Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05362 • Publication Date (Web): 06 Feb 2019 Downloaded from http://pubs.acs.org on February 6, 2019

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Analytical Chemistry

Versatile Electrochemiluminescence and Electrochemical “on-off” Assays of Methyltransferases and Aflatoxin B1 Based on a Novel Multifunctional DNA Nanotube

Junjun Ge, Yu Zhao, Chunli Li, Guifen Jie* Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science, MOE; Shandong Key Laboratory of Biochemical Analysis; Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong; College of Chemistry and Molecular Engineering. Qingdao University of Science and Technology, Qingdao 266042, PR China

ABSTRACT: Herein, a new multifunctional DNA nanotube (DNANT) was self-assembled and used to load Ru(phen)32+ and methylene blue (MB) as amplified signal probes for versatile electrochemiluminescence (ECL) and electrochemical (EC) “on-off” assays of Dam methylase (MTase) and aflatoxin B1 (AFB1). The DNA nanotube as a carrier could immobilize numerous MB or Ru(phen)32+ in the double-stranded DNA (dsDNA) to significantly amplify signals, which enabled highly sensitive ECL and electrochemical detection of dual targets. Target Dam MTase firstly catalyzed methylation of hairpin DNA (H1), then the methylated DNA was cleaved by endonuclease DpnI to expose a single strand DNA. After the Ru(phen)32+-DNANTs or MB-DNANTs signal probes were assembled to the electrode by hybridization, remarkable “signal on” state for amplified ECL or EC assays of MTase were obtained. Furthermore, in the presence of target AFB1, the structure of DNANTs was collapsed due to the specific binding of AFB1 to aptamer S2 in NTs, which led to the release of signal probes (Ru(phen)32+ or MB) from the electrode to achieve “signal off” state for dual detection of AFB1. Taking advantages of the multifunctional DNANTs amplification signal probes, the versatile biosensors showed good analytical performance with very wide linear ranges (0.001-100 U⋅mL⁻¹ and 0.0001-100 ng⋅mL⁻¹ for MTase and AFB1 assay by DPV) and lower detection 1

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limits (2.1×10-4 U⋅mL⁻¹ and 0.018 pg⋅mL⁻¹ for MTase AFB1 by DPV). This is the first time that ECL and EC “on off” methods have been achieved separately for dual targets assays, which opened a new avenue of DNANT-based signal amplification strategy for versatile design of biosensors in various biological detections. Rapidly growing number of human diseases such as cancers are associated with some chemicals such as abnormal methylase (MTase) and aflatoxin B1 (AFB1). DNA methylation of methyltransferases is abundantly present in most living organisms and regulates a variety of cellular processes.1 Abnormal DNA methylation patterns are closely related to genetic instability and the inhibition of tumor suppressor genes, which may ultimately lead to cancer.2−5 Detection of MTase activity and identification of its inhibitors are crucial to biomedical research and early cancer diagnosis. AFB1 is a popular and toxic mycotoxin that can contaminate many food products such as grain, wine, peanut, and soy products.6,7 It has been reported that exposure to AFB1 can cause severe cirrhosis, necrosis and cancer in humans and animals.8 AFB1 metabolism is mainly in the liver, but it is activated to aflatoxin-8,9-exo-epoxide in enterocytes, which react with p53 tumor suppressor gene and result in mutation at codon 249 in HCC.9 In addition, AFB1 activation allows over-expression of E2F1 protein that promotes cell growth in HCC HepG2 cells.10 Therefore, it is important to establish sensitive and reliable methods for assays of trace AFB1. DNA is becoming a powerful and versatile nanoscale building block for self-assembled nanostructures by virtue of its ideal molecular recognition property, precise nucleobase sequence control, and ease chemical functionalization.11,12 Construction of DNA nanotubes with controllable diameters is a promising and popular research branch in DNA nanotechnology, which is rapidly evolving into versatile methods for achieving subtle nanometer scale materials and molecular diagnostic devices. Various methods have been proposed to construct DNA nanotubes.13-14 DNA self-assembly provides high programmability and structural predictability.15 Helical DNA nanotubes with controllable diameters could be self-assembled,13 and applied to drug delivery by encapsulating drug cargos in the 2


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tubes.16 Helix double-strand DNA (ds-DNA) has the ability to be intercalated with some small molecule probes with high affinity,17,18 which can be used to develop DNA-related biosensors. Ru(phen)32+ complex can be intercalated into the grooves of ds-DNA and used as ECL indicator.19–21 Methylene Blue (MB) as an electrochemical probe22,23 can inserted into dsDNA through π-π stacking interactions.24 Electrochemical methods have outstanding advantages such as ease of miniaturization, simplicity, high speed and low cost. Considerable interest has been paid to developing electrochemical biosensors for rapid diagnosis of diseases.25,

26

Label-free electrochemical strategy based on MB probe and hybridization chain reaction was reported for sensitive miRNA detection.27 Electrochemiluminescence (ECL) has become a powerful analytical tool due to the advantages of low background signal, high sensitivity, simple instrument and easy control.28 Ru(phen)32+-based ECL biosensor was developed for Kras mutations by hyperbranched rolling circle amplification.29 In consideration of the above facts, DNA nanotube is ideal candidate for signal amplification in designing electrochemical and ECL biosensors for multiple targets detection. In this work, a versatile biosensor based on DNA nanotube signal amplification technology was designed for Dam MTase and aflatoxin dual targets assays by ECL and EC “on-off” detection methods. The presence of target Dam MTase induced methylation and cleavage of hairpin DNA, then the assembled DNANTs with ECL or EC signal probes (Ru(phen)32+ and MB) were linked to the electrode by hybridization, generating remarkable “signal on” state for amplified ECL or EC detection of Dam MTase. Moreover, the target AFB1 could specifically bind to aptamer S2 in DNANTs, which led to the destruction of DNANTs and release of signal probes (Ru(phen)32+ and MB) from the electrode, resulting in obvious decrease of ECL or DPV signals for AFB1 detection. The developed multifunctional DNANTs opened a new avenue of signal amplification strategy for simultaneous detection of multiple targets by versatile design with various signal probes. EXPERIMENTAL SECTION 3



Preparation of DNA nanotubes (DNANTs). The DNA nanotubes (DNANTs) were prepared according to the literature 30 with a minor modification. Three types of monomers S1, S2, and S3 (1 μM for each strand, the corresponding sequences were shown in Table 1) were used to construct the nanotubes by a one-pot annealing from 95 °C to room temperature in the Tris-EDTA (TE) buffer. Among them, S2 is an aptamer for aflatoxin B1, R1 is a single-stranded DNA that was complementary to S2 strand. After R1 was added to the solution, the DNA nanotube was constructed. Preparation of DNANTs-MB, DNANTs-Ru(phen)32+ Complex. After the DNA nanotubes were assembled, 50 μL of the products were mixed with Ru(phen)32+ (100 μL, 2 mM) under slight shaking at 37 °C for 7 h to ensure the insertion of Ru(phen)32+ into dsDNA. Similarly, 50 μL of DNA nanotubes were mixed with 100 μL of 20 mM MB with slight shaking at 37 °C for 40 min to ensure the insertion of MB into the groove of dsDNA. Thus the DNANTs-Ru(phen)32+ and DNANTs-MB complexes were successfully prepared. Construction of the ECL Biosensors. The electrodes were cleaned in ethanol and distilled water, and then scanned in 0.5 M H2SO4 from 0.2 and 1.6 V until a remarkable voltammetric peak was obtained,31 followed by sonication again and drying with nitrogen. The prepared AuNPs (20 μL) was placed on the surface of the treated gold electrode and dried naturally. Prior to use, all the hairpin DNA were heated to 95 °C for 5 min and then slowly cooled to room temperature. The SH-hairpin DNA was activated with Tris (2-carboxyethyl) phosphine (TCEP) for one hour to cleave the disulfide bond, then 10 μL of SH-hairpin DNA (1.0 μM) was placed onto the electrode and incubated at room temperature in humidity overnight. The electrode surface was rinsed with deionized water and blocked with 1 mM mercaptohexanol (MCH) for 2 h. Next, different concentrations of Dam MTase (dissolved in a mixed solution of 1×dam buffer and 80 μM SAM), 1 U/μL DpnI (dissolved in Tango buffer), and DNANTs-Ru(phen)32+ complex probe were sequentially dropped on the electrode for 2 h. Finally, different concentrations of aflatoxin B1 were dropped onto the 4


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electrode and incubated for 3 h. All of the above operations were carried out in a moist environment at 37 °C, and the electrodes were carefully rinsed with PBS buffer (pH = 7.4) after each reaction. The ECL emission was measured on a MPI-E electrochemiluminescence analyser in phosphate buffer solution (200 mM, pH 7.4) containing 20 mM TPA. The ECL signals were recorded from 0.6 to 1.6 V with the potential of the photomultiplier tube (PMT) at 600 V. Construction of the Electrochemical Biosensors. The electrochemical biosensor was constructed in the similar way with ECL, the DNANTs-MB complex probe was dropped on the electrode for 2 h. Differential pulse voltammetry (DPV) signals were measured. DPV measurements was performed on a CHI 660E workstation, and the potential was scanned in PBS from -0.4 to 0 V. 31 Assay of Methylation by Gel Electrophoresis. Before the gel electrophoresis assay, 30 µL sample 1 (1 µM hairpin DNA, 8 units of Dam MTase and 10 units DpnI, 1×dam buffer, 1×Tango buffer, 80 µM SAM)，20 µL sample 2 (1 µM hairpin DNA, 8 units of Dam MTase, 1×dam buffer, 80 µM SAM)，20 µL sample 3 (1 µM hairpin DNA, 10 units DpnI, 1×Tango buffer) were incubated at 37 °C for 3 h，respectively. The samples were then put on a polyacrylamide gel. The electrophoresis was carried in 1×tris-acetic acid-EDTA (TAE) (pH 8.0) at 180 V constant voltages for 3 min and 135 V for 1.5 h. After EB staining, the gel was scanned using the Gel imaging analyzer (Saizhi Venture Technology Co., Ltd. Beijing，China). Transmission electron microscopy (TEM) Characterization of DNA nanotubular. DNANTs need to be stained before taking TEM test. 4 μL of the prepared DNA nanotubes were dropped on the carbon-coated copper mesh for 2-3 min, then the excess reaction solution was removed with a clean filter paper. Subsequently, 2 μL of 2% uranyl acetate solution was added to the copper mesh, and excess reaction solution was removed by a clean filter paper immediately, then 2 μL of 2% uranyl acetate solution was added again. After waiting for 1 min, the excess solution was removed again. The copper mesh was then placed on clean glassware 5



and allowed to dry at room temperature. RESULTS AND DISCUSSION Principle for ECL and electrochemical detection of methylase and aflatoxin B1 based on the DNA nanotube signal probes. The design principle for versatile ECL and electrochemical “on-off” detection of Dam MTase and AFB1 based on the multifunctional DNA nanotube signal probes was shown in Scheme 1. The first part (yellow) showed the fabrication process of the multifunctional signal probes. S1, S2, S3, and R1 were firstly self-assembled into DNA nanotubes (DNANTs) by hybridization (S2 is aptamer of AFB1). Then Ru(phen)32+ as ECL signal molecules were intercalated into the groove of dsDNA to form amplified ECL signal probes. Similarly, MB molecules were combined with dsDNA by π-π stacking to form the EC probes. The second part (green) displayed the biosensor principle for ECL and electrochemical detection of methylase and aflatoxin B1 based on the DNA nanotube signal probes. The hairpin DNA was firstly linked to the Au NPs-modified electrode. Then the present target Dam MTase recognized the specific site of hairpin DNA to catalyze DNA methylation, followed by cleavage of hairpin DNA with endonuclease DpnI. Subsequently, Ru(phen)32+-DNANTs (as ECL signal probe) or MB-DNANTs (as EC signal probe) were assembled onto the electrode by DNA hybridization, thus amplified ECL or EC “signal on” detection of MTase was achieved. Moreover, when the second target AFB1 was present, it specifically bind to its aptamer S2 in NTs, which induced the destruction of DNANTs structure and release of the signal probes (Ru(phen)32+ or MB) from NTs. Then the remarkable change of ECL (or EC) signal was obtained for AFB1 detection (off state). In the absence of Dam MTase, hairpin DNA could not be methylated and cleaved, so the signal probes could not be assembled on the electrode to generate ECL or DPV signal. In the absence of AFB1, it cannot bind to S2 to destroy DNANTs, and the signal probes cannot be released to result in signal change. Therefore, sensitive “on off” detection of dual targets (Dam MTase and AFB1) can be achieved by the present ECL or DPV methods. 6


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Scheme 1. Schematic representation for construction of the DNA nanotube signal probes and versatile biosensors for ECL and electrochemical “on-off” detection of methylase and aflatoxin B1.

Characterization of Au NPs. The Au NPs were characterized by transmission electron microscopy (TEM) and UV-vis absorption spectra. Figure 1A showed the TEM image of Au NPs, good morphology and uniform particle size of Au NPs were observed, and the average diameter is about 18 nm. The inset is the particle size distribution of Au NPs, the diameter (18.16 nm) is consistent with TEM image. Figure 1B showed the UV-vis absorption spectrum of Au NPs, an absorption peak at 530 nm 7



indicates the characteristic property of Au NPs.

Figure 1. (A) TEM image of Au NPs (inset: size distribution of Au NPs). (B) UV-vis absorption of Au NPs.

Characterization of DNANTs. The prepared DNA nanotubes were characterized by TEM image. As shown in Figure 2, the DNA nanotubes are 14-46 nm in diameter, and 400 nm-2 μm in length. The resulting tubular structure demonstrate the successful formation of DNANTs.

Figure 2. TEM image of DNANTs.

In addition, the DNA nanotubes were further confirmed by atomic force microscopy (AFM) image. Figure 3A showed that the long DNA nanotubes of different lengths were successfully constructed, and the typical features of nanotubular structure were observed on mica sheets. The diameter of the DNA nanotubes is about 42.8nm, and the height is approximately 6 nm (inset). In particular, the length of DNANTs is long enough to reach micron level, which could greatly amplify probe signals in DNANTs to achieve sensitive detection of targets. Figure 3B showed the AFM image of DNA in the presence of AFB1. Since S2 in DNANTs is aptamer for AFB1, the structure of DNANTs is destroyed due to the specific binding 8


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of AFB1 to S2, and short DNA segments were observed.

Figure 3. (A) AFM image of DNANTs. (B) AFM image of DNANTs in the presence of AFB1.

PAGE Analysis of DNA Nanotubes and Methylation of Hairpin DNA. The assembled DNA nanotubes were characterized by polyacrylamide gel electrophoresis. As can be seen in Figure 4A, lane m is the marker, lanes a-c displayed the single bands at similar positions, corresponding to DNA S1, S2, and S3, respectively. The single strand DNA R1 with the lowest molecular weight run fastest, corresponding to lane d. After the single strand DNA (S1, S2, S3, and R1) were self-assembled into DNA nanotubes, DNA run slowest due to the highest molecular weight (lane e), suggesting successful formation of DNA nanotubes. After the addition of AFB1, the binding of AFB1 to aptamer S2 disrupted the structure of DNANTs, releasing a large amount of single-stranded DNA (S1, S2, S3 and R1), so several small bands were present (lane f).

Figure 4. PAGE analysis of DNA nanotubes (A) and methylation of hairpin DNA (B).

We further employed agarose gel electrophoresis to confirm the DNA 9



methylation in our assay method. As is shown in Figure 4B, when Dam MTases or Dpn I was absent, there is only one band of the original hairpin DNA (lanes 1, 2 and 3), indicating that no cleavage reaction occurs. By comparison, in the presence of Dam MTase and Dpn I, new bands appeared in lane 4, demonstrating that methylation reaction has taken place and the methylated DNA were cut into small DNA fragments. Characterization of the interaction between Ru(phen)32+ (or MB) and DNANTs. As shown in figure 5A, the fluorescence signal of Ru(phen)32+-DNANTs complex is higher than that of free Ru(phen)32+, indicating the fluorescence of Ru(phen)32+ is enhanced due to the strong intercalation between Ru(phen)32+ and dsDNA.32 The helical double-strand DNA (dsDNA) has the capacity to be intercalated with some small molecules into its grooves with high affinity.32 If this interaction is used to develop DNA-related sensors, more than one probe molecule can be intercalated into a DNA sequence without chemical modification. The results confirmed that Ru(phen)32+ was successfully inserted into the DNANTs.

Figure 5. (A) Fluorescence spectra of Ru(phen)32+-DNANTs complex. (B) UV-visible absorption spectra of MB-DNANTs complex.

The interaction between MB and DNANTs was also demonstrated by UV-visible absorption spectroscopy. As shown in figure 5B, MB has two characteristic absorption peaks at 680 and 300 nm, while DNANTs show a characteristic absorption peak at 260 nm. By comparison, three characteristic absorption peaks at 680, 300, and 260 nm for DNANTs-MB were observed, indicating that MB is successfully inserted into DNANTs through π-π stacking interactions. Herein, the difference in absorption 10


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peak intensity is caused by different concentration of MB. Characterization of the Biosensor Fabrication. The fabrication process of the DNA biosensor was characterized by cyclic voltammetry (CV). Figure S-1A displayed the voltammograms of Fe(CN)64-/3- on the different modified electrodes. Compared with the bare electrode (curve a), the peak current obviously increased on the Au NPs modified electrode due to better conductivity (curve b). After hairpin DNA and MCH were linked to the electrode, the current became smaller (curve c), as the negative DNA backbone and MCH repel [Fe(CN)6]3-/4- from the electrode. As expected, the current response increased after methylation and cleavage reaction of hairpin DNA (curve d). Then DNANTs were assembled onto the electrode, the current response decreased (curve e). Finally, the present AFB1 is specifically bound to S2 and disrupted the structure of DNANTs, the peak current significantly enhanced (curve f). EIS can give further information on the impedance changes of the modified electrode. As seen in Figure S-1B, the electron transfer resistance (Ret) changed successively in the modification process of the electrode. The results are consistent with CV, demonstrating that the biosensor was successfully fabricated. Feasibility of the electrochemical biosensor for detection of Dam MTase and AFB1. Figure 6A shows the differential pulse voltammogram of MB. It can be seen that MB displayed high electrochemical signal at a peak of -0.25V, indicating that the MB-DNANTs has good electrochemical property and great potential in chemical biosensing applications. The feasibility of the electrochemical biosensor for targets detection was investigated. As shown in Figure 6B, the bare electrode has no DPV response signal (curve a). In the absence of Dam MTase, MB-DNANTs cannot be linked to hairpin DNA on the electrode, so the DPV signal response was also very low (curve b). In the presence of Dam MTase, a relatively large electrochemical signal was observed (curve c), indicating that MB-DNANTs were successfully assembled on the electrode by methylation. Furthermore, when AFB1 was present in the reaction system, the 11



electrochemical signal showed obvious decrease (curve d), indicating that the DNANTs structure was destroyed and MB detached from the electrode surface. The results demonstrate that the present electrochemical biosensor is feasible for detection of dual targets.

Figure 6. (A) Differential pulse voltammogram (DPV) of MB, (B) DPV signals of the modified electrode under different conditions: bare gold electrode (curve a), without Dam MTase (curve b), with Dam MTase (curve c), in the presence of AFB1 (curve d).

Optimization of experimental conditions. To achieve a good experimental performance, the experimental conditions of the proposed method were optimized. The DPV signal of MB is dependent on the amount of MB molecules bound to the DNANTs, and it is also influenced by the reaction time of Dam, AFB1 and MB. As can be seen from Fig.S-2A, the DPV signal gradually increases with increasing reaction time of Dam (blue curve), and remains basically unchanged after 2 h, indicating that the optimal reaction time of Dam is 2 h. Similarly, the optimal reaction time is 2.75 h for AFB1 (Fig.S-2A, red curve) and 40 min for MB (Fig.S-2B, red curve), respectively. Fig. S-2B (blue curve) also showed that the optimal dosage for MB is 1.0 nmol. Electrochemical Biosensor for Detection of Dam MTase. Figure 7A showed the DPV signals of the assay system with increasing concentrations of target Dam MTase. Under the optimal conditions, the DPV signal increased with increasing Dam MTase concentration from 0.001-100 U⋅mL⁻¹, as more target Dam MTase could induce methylation and cleavage of more hairpin DNA, so more MB-DNANTs were assembled on the electrode to generate higher DPV signal. The DPV signal showed a 12


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good linear relationship with logarithm of Dam MTase concentration in the range of 0.001-100 U⋅mL⁻¹ (R2=0.996) (inset of Figure 7B). The detection limit (LOD, defined as 3σ/N of the signal from the blank) was 2.1×10-4 U⋅mL⁻¹, which was much lower than those in the reported detections (Table S-2). Three repeated tests for 10 U⋅mL⁻¹ Dam MTase was performed, and the relative standard deviation (RSD) was 2.24%, indicating that the biosensor has good precision, and can be applied for quantitative detection of Dam MTase concentration.

Figure 7. (A) DPV signals of the assay system with increasing concentrations of target Dam MTase (U⋅mL⁻¹). (a) 0.001, (b) 0.005, (c) 0.01, (d) 0.1, (e) 1.0, (f) 10, (g) 100. (B) Relationship between DPV peak current and concentration of Dam MTase, inset: the logarithmic calibration plot for Dam MTase detection.

Figure 8. (A) DPV signals of the assay system with increasing concentrations of target AFB1 (ng⋅mL-1). (a) 0.0001, (b) 0.001, (c) 0.01, (d) 0.1, (e) 1.0, (f) 10, (g) 100. (B) Relationship between DPV signals and concentration of AFB1, inset: the logarithmic calibration plot for AFB1 detection. The concentration of MTase is fixed at 10 U⋅mL-1.

Electrochemical Detection of AFB1. Figure 8A showed the DPV signals of the 13



assay system with increasing concentrations of target AFB1. As can be seen the DPV signal decreased with increasing AFB1 concentration from 0.0001-100 ng⋅mL-1. The specific binding of more AFB1 to aptamer S2 in NTs destroyed more DNANTs, so more MB leaved the electrode surface, leading to more decrease of DPV signal. The DPV signal vs logarithmic concentration of AFB1 showed a good linear relationship in the range of 0.0001-100 ng⋅mL⁻¹ (R2=0.998) (inset in Figure 8B). The detection limit was estimated to be 0.018 pg⋅mL⁻¹, which was much lower than those in the reported methods (Table S-3). Three parallel experiments were performed with the target of 10 ng⋅mL⁻¹, and the relative standard deviation (RSD) was 5.23%, indicating that the biosensor has good reproducibility, and can be used for detection of target AFB1 concentration. Selectivity study of the electrochemical biosensor. The selectivity of the proposed method for electrochemical detection of methyltransferase was assessed on a panel of Dam, M.SssI,33 M.CviPI34 and Alu I MTase35. In the biosensing system containing hairpin DNA, SAM and DpnI, obvious enhancement of DPV signal was observed upon addition of Dam MTase, while no any change of DPV signal was showed in the presence of M.SssI or other methyltransferases (Figure S-3A), indicating that the biosensing method has high selectivity for methyltransferase detection. The reason may originate from the fact that the interactions of MTase and DpnI with their substrates are sequence-specific.36-37 The specificity of the electrochemical biosensor for AFB1 assay was investigated by using equal amounts of AFB2, AFG1, AFG2 and mixture as interfering agents. As shown in Figure S-3B, only in the presence of AFB1 that the DPV signal was observed, and the interfering agents have no obvious effect on DPV signal, demonstrating that the electrochemical biosensor has excellent specificity for AFB1 assay. Feasibility of the ECL biosensor. Figure 9A showed the ECL intensity-potential curve of Ru(phen)32+ (inset is the ECL-time curve). It can be seen that Ru(phen)32+ displayed an ECL peak at 1.3 V and the intensity is very high, indicating that the 14


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Ru(phen)32+-DNANTs have good ECL property and show great potential in chemical biosensing applications. The reaction mechanism is as follows. TPrAH+ (DNA) → TPrA•++ H+ (DNA) + e-

(1)

TPrA•+ →(C3H7)2N(CHC2H5• ) + H+

(2)

(Ru(phen) 32+ - DNA) - e- → (Ru(phen) 33+- DNA)

(3)

(Ru(phen)33+ - DNA) + (C3H7)2N(CHC2H5•) →(Ru(phen)32+•- DNA) + [(C3H7)2N=CHC2H5]+

(4)

(Ru(phen)32+ - DNA) + (C3H7)2N(CHC2H5•) → (Ru(phen)3+ - DNA) + products

(5)

(Ru(phen)3+-DNA) +TPrA•+ → TPrA + (Ru(phen)32+*- DNA)

(6)

(Ru(phen) 32+* - DNA) → (Ru(phen) 32+ - DNA)+hυ

(7)

Figure 9. (A) ECL intensity-potential curve of Ru(phen)32+, inset: ECL intensity-time curve of Ru(phen)32+. (B) ECL intensity-time curves of the modified electrode under different conditions: (a) bare gold electrode, (b) in the absence of Dam MTase, (c) in the presence of Dam MTase, (d) in the presence of AFB1.

The feasibility of the ECL biosensor was also investigated. As shown in Figure 9B, the bare electrode showed no ECL signal (curve a). In the absence of Dam MTase, Ru(phen)32+-DNANTs cannot be linked to the hairpin DNA, so the ECL signal response is also very low (curve b). In the presence of Dam MTase, a relatively high ECL signal was observed (curve c), indicating that Ru(phen)32+-DNANTs were assembled onto the electrode. When AFB1 was present, the specific binding of AFB1 to its aptamer destroyed the DNANTs structure, thus Ru(phen)32+ detached from the electrode and led to obvious decrease of ECL signal (curve d). The above results 15



demonstrated the present ECL biosensors are feasible for assays of dual targets (Dam MTase and AFB1). Optimization of experimental condition. In order to obtain the optimum experimental conditions, the amount of Ru(phen)32+ molecules in DNANTs, the reaction time of Dam, AFB1 and Ru(phen)32+ were all investigated, which were shown in Figure S-4. As is shown in Fig.S-4A (blue curve), ECL signal gradually increased with increasing reaction time of Dam, and then became stable after 2 h, indicating that the optimal reaction time of Dam was 2 h. Similarly, the optimal reaction time for AFB1 was 2.75 h (A, red), the optimal reaction time for Ru(phen)32+ was 7 h (B, blue), and the optimal dosage for MB was 180 nmol (B, red). ECL biosensor for detection of Dam MTase. The activity of Dam MTase was detected under the optimized conditions. As can be seen from Figure 10A, the ECL intensity gradually increased with increasing Dam MTase concentration. In the presence of more Dam MTase, more hairpin DNA was methylated and cleaved to connect more Ru(phen)32+-DNANTs signal probes, generating higher ECL signal. Figure 10B displayed the calibration curve for Dam MTase assay, and a linear relationship (R2=0.995) from 0.005 to 100 U⋅mL⁻¹ was observed. A detection limit for Dam MTase is estimated to be 5×10-4 U⋅mL⁻¹ (3σ/N), which is lower than the previously reported methods (Table S-2).

Figure 10. (A) ECL response signals of the biosensor for different concentrations of Dam MTase (U⋅mL⁻¹). (a) 0.005, (b) 0.01, (c) 0.1, (d) 1.0, (e) 10, (f) 100. (B) Relationship between ΔECL （deduct background）and Dam MTase concentration, inset: the logarithmic calibration plot for Dam MTase detection. 16


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Taking 100 U⋅mL⁻¹ target Dam MTase as a model, three parallel experiments were performed. The relative standard deviation (RSD) was 2.43%, indicating that the biosensor has good precision for assay of Dam MTase, and can be used for quantitative detection of Dam MTase concentration. ECL biosensor for detection of AFB1. To investigate the analytical performance of the proposed method, the AFB1 at various concentrations were measured. Figure 11A shows the ECL response signal changes ΔECL (Difference between ECL signal with AFB1 and that without AFB1) with the concentration of AFB1. As the AFB1 concentration increased, large amounts of DNANTs were destroyed and then more Ru(phen)32+ leaved the electrode surface, resulting in corresponding decrease of ECL intensity. Figure 11B showed the relationship between the ΔECL intensity and AFB1 concentration, and a logarithmic calibration curve for AFB1 detection from 0.0001 to 100 ng⋅mL⁻¹ was obtained (inset, R2=0.996). The detection limit for AFB1 is estimated to be 0.058 pg⋅mL⁻¹ (3σ/N), which is lower than the previously reported methods (Table S-3). By performing three times detection on the target at 10 ng⋅mL⁻¹, the relative standard deviation (RSD) was calculated to be 3.45%, indicating that the method has good reproducibility for AFB1 assay, and can be used for quantitative detection of AFB1 concentration.

Figure 11. (A) ECL response signal changes of the assay system in the presence of increasing concentrations target AFB1 (ng⋅mL⁻¹). (a) 0.0001, (b) 0.001, (c) 0.01, (d) 0.1, (e) 1.0, (f) 10, (g) 100. (B) Relation curve of the assay system for target AFB1 detection, inset: calibration plot for AFB1 detection. The concentration of MTase is fixed at 100 U⋅mL-1.

Selectivity study of the ECL biosensor. To investigate the selectivity of the 17



proposed ECL method for Dam MTase assay, M.SssI, M.CviPI and Alu I MTase as interference enzyme were introduced. Due to the specific site recognition of Dam MTase toward its substrate, the proposed method can easily discriminate Dam MTase from other interference MTase. As shown in Figure S-5A, significant ECL enhancement is observed in the presence of Dam MTase. In contrast, no distinct ECL signal is observed in the presence of M.SssI, M.CviPI and Alu I MTase, suggesting the proposed method possess high selectivity toward Dam MTase assay. The selectivity of the ECL biosensor for AFB1 against the interfering substances such as AFB2, AFG1, AFG2 was studied (Figure S-5B). It can be seen that the nontarget analytes including AFG1, AFG2 and AFB2 did not cause obvious change in ECL intensity, which is similar to the background signal (blank sample). While the target AFB1 is present (AFB1 and mix), the ECL intensity obviously decreased due to the specific binding of AFB1 to its aptamer in DNANTs, suggesting that the ECL biosensor has high selectivity for AFB1 assay. Application of the proposed method in real sample analysis. To evaluate the potential application of the proposed method, the electrochemical biosensor was used for detecting peanut samples by standard addition method. The samples were spiked with standard AFB1 at 0.01, 0.005, and 0.002 ng⋅mL⁻¹ for the recovery test, respectively. The accuracy of the results was evaluated by relative standard deviation (RSD). No AFB1 was found in the clean samples (Figure S-6A), while AFB1 was present in all moldy peanut samples (Figure S-6B). The measured results were displayed in Table S-4, the recovery and RSDs were in the range of 97.0-107.8% and 3.8-5.4%, indicating that the electrochemical biosensor has good accuracy and is acceptable for food sample analysis. CONCLUSIONS In this work, versatile biosensors based on the DNA nanostructure signal amplification technology for dual targets assays of Dam MTase and AFB1 were presented by ECL and EC detection methods. The self-assembled DNA nanotubes are introduced to load a large number of Ru(phen)32+ and MB as amplified ECL and EC 18


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signal probes for versatile detection of dual targets. The presence of target Dam MTase induced methylation and cleavage of hairpin DNA, which enabled Ru(phen)32+-DNANTs or MB-DNANTs signal probes to be assembled onto the electrode to achieve “signal on” state for amplified ECL or EC assays of Dam MTase. The present AFB1 led to collapse of DNA nanotubular structure due to the specific binding of AFB1 to aptamer, which achieved “signal off” detection of AFB1 by releasing signal probes. It is for the first time that the novel DNANTs were designed as signal amplification strategy for four kinds of detection in a system, the proposed biosensors can achieve “on” to “off” assays of diverse targets with wide linear ranges and extremely low detection limits. In view of these advantages, this work would open a promising avenue to develop highly sensitive versatile sensing systems using DNANTs amplification platform for various biological applications and early disease diagnosis.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 21575072). ASSOCIATED CONTENT Supporting Information Reagents and instruments, DNA sequences, preparation of gold nanoparticles, extraction of AFB1 from peanut samples, characterization of the electrodes, optimization of experimental conditions, selectivity of the biosensors, comparison of the proposed methods with some reported methods, real samples detection. REFERENCES (1) Chen, S.; Lv, Y.; Shen, Y.; Ji, J.; Zhou, Q.; Liu, S.; Zhang, Y. Highly Sensitive an (2) d Quality Self-Testable Electrochemiluminescence Assay of DNA Methyltransferase Activity 19



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