Electro-Grafted Electrode with Graphene-Oxide-Like DNA Affinity for

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Electro-Grafted Electrode with Graphene-oxide-Like DNA Affinity for Ratiometric Homogeneous Electrochemical Biosensing of MicroRNA Lei Ge, Wenxiao Wang, and Feng Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02896 • Publication Date (Web): 10 Oct 2017 Downloaded from http://pubs.acs.org on October 11, 2017

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Analytical Chemistry

Electro-Grafted Electrode with Graphene-oxide-Like DNA Affinity for Ratiometric Homogeneous Electrochemical Biosensing of MicroRNA

Lei Ge, Wenxiao Wang and Feng Li*

College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao, 266109, People’s Republic of China

*Corresponding author: Feng Li E-mail: [email protected] Telephone: +86-532-86080855

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ABSTRACT This work demonstrated for the first time a simple and rapid approach to endow the electrode with the excellent discrimination ability over single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) through the robust electrochemical grafting of in-situ generated 1-naphthalenesulfonate (NS–) diazonium salt onto the surface of indium tin oxide (ITO) electrode. On the basis of understanding the influence of sequence and length on the binding affinity of ssDNA and dsDNA toward NS– grafted ITO (NS–-ITO) electrode, these interesting findings were successfully employed to rationally develop a ratiometric homogeneous electrochemical biosensing platform for microRNA based on the affinity-mediated signal transduction. The achievement of ultrasensitive detection of microRNA lies in a compatibly designed T7 exonuclease-assisted isothermal amplification strategy, in which the presence of target microRNA initiated the continual and opposite affinity-inversion of two rationally engineered electrochemical signal reporters, methylene blue (MB) labeled hairpin reporter and ferrocene (Fc) labeled dsDNA reporter, toward NS–-ITO electrode, thereby providing the ratiometric transduction and amplification of the homogeneous electrochemical output signal. By measuring the distinct variation in the peak current intensity ratios of Fc and MB tags, this ratiometric homogeneous electrochemical microRNA biosensing platform showed a detection limit of 25 aM, which is much lower than that of the reported homogeneous electrochemical biosensors. Therefore, we envision that the proposed approach will find useful applications in disease molecular diagnoses and biomedicine.


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INTRODUCTION MicroRNA (miRNA) are a promising class of biomarker candidates for clinical cancer diagnosis, prognosis, and therapy in asymptomatic individuals.1-6 Therefore, detection and quantitation of the miRNA expression level in biological samples or clinical specimens is thus of great importance to cancer research and diagnosis.7-10 Moreover, because of their small size, sequence homology among family members, and low level in body fluids,11 novel analytical methods for the detection of extremely trace miRNA are being actively pursued. Among various protocols for the sensitive and selective determination of miRNA,12-22 electrochemical techniques are frequently employed as rapid and still efficient analytical protocols,23-27 which not only require relatively simple instruments and operations but also allow for instrument miniaturization and portability. Recently, homogeneous electrochemical strategies have earned them great attention as a novel powerful bioanalytical tool28-34 because of their independence of expensive and complicated surface-immobilization of biorecognition-probe, which offered new opportunities towards applications in electrochemical biosensors for sensitive biological analysis. Compared with the conventional electrochemical methods, the target-recognition reactions as well as the signal-amplification reactions for homogeneous electrochemical strategies35-37 occur in the homogeneous solution,38-40 instead of on the surface of electrodes and/or (nano-)materials, which could effectively avoid the bio-recognition probe’s geometry change induced by the steric hindrance effect on the electrode/(nano-)material surface41, 42 and, thus, maximally maintain their configurational freedom, not only



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improving their recognition efficiency and rate towards targets, but also keeping them away from the chemical impairment of their bioactivity. However, all the reported homogeneous electrochemical biosensors only offer the change of single peak current intensity as the electrochemical signal output, which is easily influenced by unavoidable internal/external disturbance induced by instrumental or environmental conditions,43-47 leading to inaccurate quantification. In contrast to the types of single-current-intensity measurements, ratiometric dual-current-signal responses can provide self-calibration to minimize the experimentally and environmentally dependent factors/interferences and exhibit strong anti-interference ability,48-51 which are favorable for more effective miRNA detection in biological environment. Unfortunately, no report has been made on the construction of ratiometric homogeneous electrochemical biosensing platform until now. To address this issue and as a continuation of our researches,52-54 we demonstrated, herein, a novel homogeneous electrochemical biosensing platform with the ratio of dual current-signaling response as signal readout through combining the features of the affinity-mediated homogeneous electrochemical signal transduction method.55-58 Recent studies have demonstrated that graphene oxide (GO) shows preferential binding to single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA).59-61 Although this fascinating property of GO has facilitated its application as electrode interface in promising DNA-based electrochemical55-58 biosensing platforms for the detection of various targets, the preparation and/or immobilization of GO on electrode


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surface is relatively complicated and time-consuming. Therefore, on the basis of the interaction mechanism between DNA and GO,62-65 we demonstrate an alternative approach to endow the electrode with the effective discrimination ability over ssDNA and dsDNA through the simple, rapid, and robust electrochemical grafting of in-situ generated 1-naphthalenesulfonate diazonium salt onto the surface of electrode, the concept of which was schematically shown in Figure 1A. We found that, similar to conventional graphene-based homogeneous electrochemical electrode,55-58 the generated thin layer of 1-naphthalenesulfonate anion shows much higher affinity toward non-structured ssDNA due to the strong π−π stacking interaction between the ring structures in nucleobases of ssDNA and the bi-hexagonal cells of naphthalene, while its interaction with dsDNA, which effectively shields its nucleobases in the helical structure, is disfavored since both of them are negatively charged. Then, for the achievement of homogeneous electrochemical miRNA biosensing with high sensitivity and accuracy, in this work, a novel T7 exonuclease (T7-Exo)-assisted isothermal amplification strategy was compatibly designed based on the target-induced affinity inversion of two rationally engineered signal reporters, methylene blue (MB) labeled-hairpin reporter and ferrocene (Fc) labeled-dsDNA reporter, toward electrode, realizing the ratiometric transduction and amplification of the homogeneous electrochemical output signal. Finally, good recovery of target miRNA in human serum samples was obtained, indicating that this ratiometric homogeneous electrochemical strategy will provide a great potential for the development of an ultrasensitive biosensing platform for medical research and early



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clinical diagnostics.

EXPERIMENTAL SECTION Reagents. All synthetic DNA and RNA sequences (illustrated in supporting information) and T7-Exo were purchased from Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, china). All sequences were HPLC-purified and freeze-dried by the supplier. They were used as provided and diluted in 10 mM sterile phosphate-buffered saline (PBS, pH 7.4) to a concentration of 100 µM. Sodium 4-amino-1-naphthalenesulfonate (NaANS) and NaNO2 were purchased from Sigma-Aldrich. Indium tin oxide (ITO) slices were ordered from China Southern Glass Holding Co., Ltd. All the chemicals used were of analytical reagent grade and used without further purification. All buffers and aqueous solutions were prepared with ultrapure water (Milli-Q, Millipore, resistance > 18.2 MΩ). Apparatus. All electrochemical experiments, including cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV), were carried out on an Autolab electrochemical workstation (Metrohm,

The

Netherlands)

at

room

temperature

using

a

conventional

three-electrode system with an modified ITO working electrode, a Pt wire counter electrode, and an Ag/AgCl reference electrode. The ITO electrode was cleaned by ultrasonic rinsing in acetone, ethanol and water, respectively, for 30 min each, followed by washing copiously with ultrapure water and dried under a stream of N2 gas. X-ray photoelectron spectroscopy (XPS) was recorded on an ESCALAB 250Xi spectrometer (Thermo Fisher, Waltham, MA) with a monochromatic Al Kα X-ray


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source (1486.6 eV) operating at a pressure of 10−9 mbar. Fabrication of NS– grafted ITO electrode. First, 1-naphthalenesulfonate anion (NS–) diazonium salt was synthesized according to our previous work. Briefly, NaANS (final concentration: 2 mM) and NaNO2 (final concentration: 5 mM) were dissolved in an aqueous solution of 0.5 M HCl. The mixture was then degassed with nitrogen flow and left to react for about 30 min at ice-bath. Second, NS–modified ITO surface were prepared by electro-grafting of NS– onto cleaned ITO using the solution of NS– diazonium salt, which was carried out by four consecutive CV cycles in potential range between 0 V and -0.5 V versus Ag/AgCl at a scan rate of 100 mV/s. After electric-grafting, the obtained ITO electrode was ultrasonic treatment in acetone, ethanol, and ultrapure water for 5 min, respectively, and finally dried by nitrogen blow. Ratiometric homogeneous electrochemical detection of miRNA. First of all, The Fc-labeled ssDNA was hybridized with its complementary DNA to form a duplex structure. The MB-labeled hairpin oligonucleotide was refolded into hairpin structures through annealing at 95 °C for 3 min and then allowed to gradually cool to room temperature. The experiments were performed in 100 µL of PBS solution containing 2.0 µM MB-labeled hairpin, 5.0 µM Fc-labeled dsDNA, 0.1 U/µL T7-Exo, and target miRNA at different concentrations. The mixture solutions were first incubated at 37 o

C for 60 min. Next, the mixture solutions were transfer to the modified ITO electrode

surface and incubated at room temperature for 30 min, followed by thoroughly rinsing with 10 mM pH 7.4 PBS. Finally, the DPV responses of the obtained electrodes were



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recorded in 10 mM pH 7.4 PBS with the potential window from –0.4 V to +0.6 V (vs Ag/AgCl). All DPV curves are baseline-corrected using the Nova 1.1 software embedded in the Autolab electrochemical workstation.

RESULTS AND DISCUSSION Typically, as shown in Figure 1A, the electrochemical grafting of in-situ generated NS– diazonium salt takes place in two steps: (i) the NS– diazonium salt is generated in situ from the reaction between the ANS– precursor and NaNO2 in the presence of HCl and (ii) NS– diazonium salt is then reduced by taking one electron from the ITO electrode and producing active free naphthyl radicals, which subsequently bond covalently to the surface of ITO electrode (NS–-ITO). Figure 1B shows the recorded typical CVs of electrochemical grafting of NS– group onto the ITO electrode surface. The first cycle of grafting CV scans shows an irreversible reduction peak at a potential of approximately −0.4 V (vs Ag/AgCl). This is attributed to the electrochemical reduction of diazonium salts at the ITO electrode surface, leading to the generation of reactive naphthyl radicals. The electro-generated naphthyl radical then reacts with the ITO electrode surface and forms a covalent bond between the NS– and the ITO surface. It is noticed that the reduction peak current is remarkably attenuated in subsequent cycles. Such a phenomenon can be assigned to the formation of the self-limiting NS– thin layer on ITO electrode during the grafting process, which can not be observed using SEM characterization (Figure S1) and, thus, was further confirmed by the XPS measurement (Figure S2). After four CV cycles, however, the electrochemical reduction peak is still observable, indicating a slow


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passivation process during electro-grafting of NS– diazonium salts on ITO electrode surface.

Figure 1. (A) Schematic illustration of in situ diazonium salt generation and their electroreduction onto the surface of ITO electrode; (B) The recorded CV curves during the electrografting of NS–; (C) The recorded CV curves of ITO electrode in the same electrolyte solution without the addition of ANS– (bottom) or NaNO2 (top) under identical conditions; (D) Nyquist plot of (a) bare ITO, (b) NS–-ITO, (c) NS–-ITO after MB-A20 adsorption, and (d) NS–-ITO after MB-A20/T20 adsorption. EIS measurement were performed in 5 mM [Fe(CN)6]3−/4− solution containing 0.1 M KCl.

In order to confirm that the electrochemical reduction peak is due to the electro-grafting of the free NS– diazonium salts onto ITO surface, The electro-grafting CV scans were recorded in the same electrolyte solution without the addition of ANS– or NaNO2 under identical conditions, and the results are illustrated in Figure 1C. As expected, no reduction peak is observed in both electrolyte solutions, indicating that the NS– groups can not be grafted onto the ITO surface in the absence of NaNO2 or ANS– under the same experimental conditions, which is consistent with the XPS result in Figure S2B. To further confirm the surface modification of ITO electrode, we then studied the EIS of the modified ITO electrodes with different interface properties using [Fe(CN)6]3-/[Fe(CN)6]4- redox probes, and the typical results were recorded in Figure 1D in the form of a Nyquist plot, in which the diameter of the semicircle at



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higher frequencies is associated with the resistance of charge transfer (Ret), and the linear part at lower frequencies relates to the diffusion process. As shown in Figure 1D, bare ITO electrode exhibits a small semicircular domain (curve a), indicating a low Ret and illustrating a characteristic of diffusion process. After the electro-grafting of NS– on the ITO surface, diameter of the semicircle part increases significantly (curve b) due to the formation of a negatively charged interface, which may severely hinder the diffusion of negatively charged [Fe(CN)6]3-/[Fe(CN)6]4- redox probes toward the ITO electrode surface. All above results confirmed the fact that the NS– thin layer was electro-grafted on the ITO surface as expected.


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Figure 2. (A, B, C, D) DPV peak current intensity changes observed after incubation of the NS– -ITO with MB-labeled ssDNAs (red/blue dots) or their corresponding dsDNAs (black dots) for different time; (A1, B1, C1, D1) DPV responses of NS–-ITO toward MB-labeled ssDNAs (red/blue lines) or their corresponding dsDNAs (black lines) after incubation for 30 min; (A2, B2, C2, D2) Illustration of the used DNA sequences. The concentrations of all DNAs were fixed at 1.5 µM.

The basis of this work is the differential affinity of NS–-ITO toward ssDNA versus dsDNA. To demonstrate this property, typically, MB-labeled homopolymer strands of adenine (A), thymine (T), or cytosine (C) bases with DNA lengths of 20-mer as well as their dsDNA counterpart (Table 1S) were employed to investigate the interaction of DNA with NS–-ITO. Because of the extreme difficulties associated with the synthesis of long homopolymer strands of guanine (G) bases (>6-mer),



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copolymer strands of G-T or A-G bases with the same base number were employed to test the affinity of NS–-ITO toward ssDNAs containing G bases. Obviously, none of the employed DNA strands can form stable secondary structures under experimental conditions. Adsorption of the MB-labeled DNA onto the surface of NS–-ITO could bring the MB tag close to electrode surface, generating the DPV response of MB tag. Therefore, the DPV peak current intensity is directly proportional to the amount of DNA adsorbed. Upon incubation with NS–-ITO, as shown in Figure 2, both MB-labeled ssDNA (red and blue dots/lines) and their dsDNA counterpart (black dots/lines) exhibited an obvious DPV response. For example, as shown in Figure 2A and 2A1, a DPV peak current of 953 nA could be obtained within 30 min for MB-labeled poly(dA)20 (MB-A20) ssDNA, while only 75 nA was acquired for its dsDNA counterpart (MB-A20/T20), which is similar with previous reports showing that both ssDNA and dsDNA could be adsorbed onto GO sheet and ssDNA exhibits a stronger or faster adsorption.66 Moreover, the results show that, for instance, the DPV response of MB-A20 is approximately 13-fold higher than that of MB-A20/T20 (Figure 2A1), surpassing two typical water-soluble anionic diazonium salts (Figure S3A1 and Figure S4A1) under the same condition, indicating that the proposed NS–-ITO is more competent for the discrimination of ssDNA and dsDNA. Furthermore, the negligible DPV responses (Figure S5) obtained from the adsorption of MB-ssDNAs and MB-dsDNAs on bare ITO electrode indicated that the thin layer of NS– grafted on ITO electrode plays key roles in the adsorption and discrimination of ssDNA and dsDNA.


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The differential affinity of NS–-ITO toward ssDNA versus dsDNA can be further confirmed by the EIS measurement. After the adsorption of MB-A20 on the modified ITO electrode, as shown in Figure 1D, a remarkable increase in Ret was observed (curve c), which can be ascribed to the repellence to [Fe(CN)6]3-/[Fe(CN)6]4- redox probes from negative charges on the phosphate backbone of the adsorbed ssDNA strands. In contrast, the adsorption of dsDNA strands only leaded to a slight increase in the Ret of modified ITO electrode (curve d), confirming the higher affinity of NS– -ITO toward ssDNA than dsDNA. Moreover, the binding strength of interactions between nucleobases and NS–-ITO can be clearly observed from peak current intensity in Figure 2, which varies in the order A ≈ C > G > T. Next, we studied the length-dependent DNA binding to NS–-ITO electrode surfaces to provide further insights for the design of ratiometric homogeneous electrochemical biosensing platform. As shown in Figure S6A, ssDNAs with lengths of 20-mer exhibit the maximal peak current intensity, suggesting the strongest binding affinity. We noticed that the peak current intensity was lower for the shorter or longer ssDNAs, which is consistent with previous reports showing a similar trend on GO.62, 64, 67 In contrast, the binding affinities of all dsDNAs were observed to be very weak (Figure S6B). Moreover, the number of scanning cycles for electro-grafting of NS– was also optimized (Figure S7) to be four cycles.



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Scheme 1. Schematic illustration of the design principle for the amplified ratiometric homogeneous electrochemical biosensing of miRNA on the NS–-ITO electrode. The sequence of DNA molecules were described in terms of encoded domains, each of which represents a short fragment of DNA sequence. Complementarity between encoded domains is denoted by an asterisk.

After confirming the discrimination ability of NS–-ITO electrode toward ssDNA versus dsDNA, we reason that this modified ITO electrode could serve as a homogeneous electrochemical biosensing platform for quantitative miRNA analysis. Scheme 1 outlines the design principle for the amplified ratiometric homogeneous electrochemical analysis of miRNA on the NS–-ITO electrode. Further support that verifies the activation of the T7-Exo-assisted isothermal amplification reaction was obtained by nondenaturating polyacrylamide gel-electrophoresis experiments (Figure 3). As shown in Scheme 1, this biosensing platform features two rationally cascaded digestion-amplification cycles by T7-Exo, which employed two ingeniously designed electrochemical probes as signal reporters, i.e. a hairpin DNA reporter and a long ssDNA reporter. The hairpin reporter is modified near its 5’-end with an electroactive MB tag, which is denoted as MB-HR in this work, and contains two important sequence domains: a 5’-overhang red fragment (domain-x*, Scheme 1) that exhibit


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partial complementarity to the target miRNA sequence as the target miRNA recognition element and a green fragment at its 3’-end (domains-a,b,c, Scheme 1) that acts as the primer sequence to initiate the next digestion cycle through toehold-mediated strand-displacement reaction. As shown in Scheme 1, the MB-HR self-hybridizes into a stable stem-loop structure with a large ssDNA loop (20 bases of adenine−cytosine−rich sequence) and a long 5’-ssDNA overhang (18 bases), and shows a single and narrow electrophoresis band (Figure 3, lane-b). As the adsorption of single-stranded loop and overhang on the surface of NS–-ITO electrode effectively makes the MB tags close to the electrode surface, the MB-HR could afford a high DPV response at about −0.25 V (vs Ag/AgCl) in the absence of miRNA (Figure 4, curve a). In contrast, as shown in Scheme 1, the binding of the Fc-labeled ssDNA (Fc-ssDNA, lane-c in Figure 3) to the electrode surface is effectively blocked through the hybridization with its complementary ssDNA sequence (helper ssDNA, lane-d in Figure 3), forming a stable duplex structure (16 base pairs), which make the formed Fc-labeled duplex DNA reporter (Fc-DR, lane-e in Figure 3) difficult to bind to the surface of NS–-ITO electrode, guaranteeing a low DPV response of the Fc tag at about +0.38 V (vs Ag/AgCl) in the absence of target miRNA (Figure 4, curve b). In the absence of both miRNA target and T7-Exo, the mixture of MB-HR and Fc-DR shows negligible peak current change for both MB-tag and Fc-tag (Figure 4, curve c), which could be ascribed to the low level of spontaneous interactions between MB-HR and Fc-DR in the absence of miRNA target (Figure 3, lane-f). Upon T7-Exo introduction, the lack of any obvious peak current change (Figure 4, curve d) as well as any



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obvious electrophoresis band shift (Figure 3, lane-g) in the absence of target miRNA indicates that the both MB-HR and Fc-DR structure are resistant to the digestion by T7-Exo. These results demonstrated .the low background signal leakage of this amplification system in the absence of target.

Figure 3. Native polyacrylamide gel electrophoresis confirmation of the ratiometric T7-Exo-assisted isothermal amplification reaction. Lane-a, let-7a miRNA; lane-b, MB-HR; lane-c, Fc-ssDNA; lane-d, helper ssDNA; lane-e, Fc-DR; lane-f, MB-HR + Fc-DR; lane-g, MB-HR + Fc-DR + T7-Exo; lane-h, MB-HR + let-7a; lane-i, MB-HR + let-7a + T7-Exo; lane-j, synthetic primer; lane-k, synthetic primer + Fc-DR; lane-l, MB-HR + Fc-DR + let-7a + T7-Exo.

Figure 4. DPV response of NS–-ITO toward MB-HR (a); Fc-DR (b); MB-HR + Fc-DR (c); MB-HR + Fc-DR + T7-Exo (d); 0.5 fM miRNA + MB-HR +Fc-DR + T7-Exo (e); 20 fM miRNA + MB-HR +Fc-DR + T7-Exo (f). The concentration of MB-HR, Fc-DR, and T7-Exo was 2.0 µM, 5.0 µM, and 0.1 U/µL, respectively.

When the biosensing platform was subjected with target miRNA, the recognition of target miRNA with the domain-x* of MB-HR formed an MB-HR@miRNA complex (the new band in lane-h, Figure 3) with blunt 5’-terminus. Since the


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5’-termini of MB-HR are labeled with the MB tag, in the presence of the T7-Exo, the blunt 5’-ends of the MB-HR@miRNA complex was digested, leading to the release of the ssDNA primer, the green region of MB-HR (Scheme 1), to the solution and to the generation of MB-labeled mononucleotides (MB-mNs), which remarkably declines the DPV response of MB tag at about −0.25 V (vs Ag/AgCl, Figure 4, curve e) due to the weak affinity of MB-mNs to the surface of NS–-ITO electrode. As illustrated in Figure 3, the generated new band, appearing at the minimum electrophoresis distance in lane-i, corresponds to the ssDNA primer (lane-j). Domain-a of the released ssDNA primer hybridized to the domain-a* at the 5’-end of Fc-DR as toehold and, then, dissociated Fc-ssDNA through the toehold-initiated branch migration reaction (lane-k, Figure 3). The formation of the primer/helper duplex (the new band with the slowest gel-shift mobility in lane-k, Figure 3) triggers the selective enzymatic hydrolyzation of helper ssDNA, the orange sequence in Scheme 1, by T7-Exo, resulting in the recycling of the intact primer sequence (lane-l, Figure 3), which is able to hybridize with a new, undigested Fc-DR and catalyze a new cycle of Fc-DR transformation. This accordingly results in the abundant accumulation of Fc-ssDNAs, which possess strong affinity toward the NS–-ITO electrode surface, leading to a distinct increase in the DPV response of Fc tag at about +0.38 V (vs Ag/AgCl, Figure 4, curve e). Furthermore, accompanied with the digestion process in recycling-I, the target miRNA was also released (lane-i and lane-l, Figure 3), which can then hybridize with another MB-HR to initiate the next round of cascaded probes digestion. In this way, a single target miRNA is able to initiate the continual generation of numerous MB-mNs



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as well as Fc-ssDNAs. Moreover, the opposite peak current changes of two labeled tags with the increase of the target miRNA concentration (Figure 4, curve f) further validated the cascaded transformation/digestion of MB-HR and Fc-DR. By measuring the distinct variation in the peak current intensity ratios of Fc and MB tags (RFc/MB), this strategy is expected to provide a novel ratiometric path for the amplified homogeneous electrochemical detection of miRNA. To confirm the ability of the proposed strategy to sensitively detect target miRNA, as a proof-of-concept, a series of different concentrations of let-7a miRNA were measured under the optimal condition. Figure 5A depicts the resulting DPV response of this ratiometric homogeneous electrochemical biosensor subjected to various let-7a miRNA concentrations. Upon treatment of the proposed biosensing system with increasing let-7a miRNA concentrations, more MB-HR could be digested and a gradual reduction in the DPV current of MB tag (Figure 5A) was observed. In contrast, the peak current of Fc tag intensified remarkably with the increase of target miRNA concentration (Figure 5A), which was ascribed to the fast generation of the Fc-ssDNA. Concomitantly, significant variations in RFc/MB were observed when the concentration of let-7a increased. Figure 5B displays the relationship between RFc/MB and let-7a concentrations. The logarithmic (lg) value of RFc/MB was found to be linearly related to the logarithm of target miRNA concentration in a range from 80 aM to 300 fM. The regression equation was lgRFc/MB = 0.3433×lg[clet-7a(fM)]−0.055 with a linear correlation coefficient of 0.9973 (n = 6) and a detection limit of 25 aM at 3σ. Such biosensing platform achieved an impressive sensitivity compared to other


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existing assay techniques and is by far one of the most sensitive methods among the homogeneous electrochemical biosensing systems for the detection of DNA/RNA (Table S2).

Figure 5. (A) DPV responses of the proposed ratiometric homogeneous electrochemical biosensing platform to different concentrations of target miRNA (from a to j: 0, 0.08, 0.2, 0.5, 2.0, 8.0, 20, 60, 120, and 300 fM). (B) The linear relationship of the logarithmic value of RFc/MB versus the logarithm of let-7a concentration. (C) Selectivity investigation of the propose ratiometric homogeneous electrochemical biosensing platform using the let-7 miRNA family as a model system. The concentration of let-7a is 0.5 fM. The concentration of each let-7b,c,d,e,f,g is 20 fM.

In order to evaluate the specificity of the proposed ratiometric homogeneous electrochemical biosensor, we performed a series of contrast experiments to detect other six members of the let-7 miRNA family (let-7b, let-7c, let-7d, let-7e, let-7f, let-7g), which differ by only one or two nucleotides in sequence with the same length (only 22 bases), using the let-7a-specific MB-HR. As shown in Figure 5C, the RFc/MB of 0.5 fM let-7a target miRNA is much higher than that produced by 20 fM negative controls. The measured data demonstrated that the proposed biosensing platform possesses the competence to distinguish the concomitant miRNA family members with sequence homology. A great challenge of an excellent miRNA assay is its ability to be applied in complex biological matrixes. The feasibility of the proposed ratiometric homogeneous electrochemical biosensor for miRNA detection in complex biological matrixes was



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demonstrated by determining the recovery of target let-7a miRNA spiked in 10% diluted human serum samples. The analytical results are shown in Table 1. When spiking with 0, 0.5, 5.0, and 50 fM target miRNA, the measured average recoveries for six determinations were 94.5%, 106.2%, and 96.1%, respectively. These results clearly indicated that the proposed ratiometric homogeneous electrochemical biosensing platform shows potential for application in miRNA detection in the human serum samples with acceptable accuracy and reliability. Table 1. Recoveries of the proposed biosensing platform for let-7a spiked human serum samples Samples Sample-1 Sample-2 Sample-3 Sample-4

Spiked (fM) 0.0 0.5 5.0 50

Measured (fM) a

– 0.473±0.035 5.31±0.27 49.3±2.16

Recovery (%)

–a 94.5 106.2 96.1

a

“−” denotes that the concentration of target miRNA is too low to be detected.

CONCLUSION In summary, we have developed a novel affinity-mediated homogeneous electrochemical biosensing platform for miRNA quantification with the ratio of dual current-signaling response as signal readout on the NS–-ITO electrode, which is capable of binding to ssDNA with a high affinity while its affinity for dsDNA is much lower, eliminating the requirements of complicated preparation/immobilization of GO on the surface of electrode. Meanwhile, we found that the binding between DNA and NS–-ITO electrode was strongly affected by both length and sequence of DNA, which formed one basis of the ratiometric homogeneous electrochemical miRNA biosensing platform. The ultrasensitive detection of miRNA relies on the T7-Exo enzymatic digestion reaction and target recycling to initiate MB and Fc assisted ratiometric signal transduction and signal amplification. To the best of our knowledge, the


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development of a ratiometric homogenous electrochemical system has never been reported before. Moreover, this ratiometric homogenous electrochemical biosensing platform exhibited high detection sensitivity toward let-7a miRNA with a detection limit down to aM level and allowed effective discriminating among miRNA family members. It also displayed acceptable recovery toward target miRNA in the real human serum samples, illustrating good feasibility for real sample detection. Furthermore, the excellent discrimination ability of NS–-ITO toward ssDNA and dsDNA suggests NS–-ITO as a versatile and universal platform appropriate for future advances in research on affinity-mediated homogeneous electrochemical biosensing. On the basis of the distinctive properties presented above, we expect that this study could broaden the perspective of homogeneous electrochemical approach for further development of ultrasensitive biosensing platform.

ACKNOWLEDGEMENTS This work was financially supported by National Natural Science Foundation of China (31501570 and 21575074), Natural Science Foundation of Shandong Province, China (ZR2014BQ011), Basic Research Program of Qingdao (16-5-1-55-jch), Research Foundation for Distinguished Scholars of Qingdao Agricultural University (663-1115003 and 663-1113311), and the Special Foundation for Distinguished Taishan Scholar of Shandong Province (ts201511052).

ASSOCIATED CONTENT Supporting Information Sequences of oligonucleotides used in this work, Figure S1-S7 as described in



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the text, and comparison of the present study with other biosensors. This material is available free of charge via the Internet at http://pubs.acs.org.

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For TOC only


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Electro-Grafted Electrode with Graphene-Oxide-Like DNA Affinity for

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