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Paper-based closed Au-Bipolar electrode electrochemiluminescence sensing platform for the detection of miRNA-155
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Paper-based closed Au-Bipolar electrode electrochemiluminescence sensing platform for the detection of miRNA-155 Fangfang Wang a, 1, Cuiping Fu a, 1, Chuan Huang a, 1, Na Li b, Yanhu Wang c, **, Shenguang Ge a, *, Jinghua Yu b a
Collaborative Innovation Center of Technology and Equipment for Biological Diagnosis and Therapy in Universities of Shandong, Institute for Advanced Interdisciplinary Research, University of Jinan, Jinan, 250022, PR China School of Chemistry and Chemical Engineering, University of Jinan, Jinan, 250022, PR China c Shandong Key Laboratory of TCM Quality Control Technology, Shandong Analysis and Test Center, Qilu University of Technology (Shandong Academy of Sciences), Jinan, 250014, PR China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Electrochemiluminescence Bipolar electrode MicroRNA-155 AuPd nanoparticles.
This paper introduces a paper-based closed Au-bipolar electrode (BPE) biosensing system for the rapid and sensitive electrochemiluminescence (ECL) detection of miRNA-155. This microfluidic paper-based sensing platform is formed by wax-printing technology, screen printing method and in-situ Au nanoparticles (NPs) growth to form hydrophilic cells, hydrophobic boundaries, water proof electronic bridge, driving electrode re gions and bipolar electrode regions. For rapid and sensitive detection, the cathode of bipolar electrode was modified with the prepared DNA (S1)–AuPd NPs by hybridization chain reaction, in which the target could initiate multiple cycles reaction to load more AuPd NPs which catalyzed H2O2 reduction. In addition, a classical ECL system tris (2,20 -bipyridine) ruthenium (II)- tripropylamine (Ru(bpy)2þ 3 /TPrA) exists at the anode of the bipolar electrode. Due to the charge balance between the anode and the cathode of BPE, the ECL signal response of Ru(bpy)2þ 3 /TPrA system was enhanced in the reporting cell. The intensity of ECL was quantitatively correlated with the concentration of miRNA-155 in the range of 1 pM–10 μM with the detection limit 0.67 pM. Moreover, this method paves a novel way for highly sensitive detection of miRNA-155 in clinical application.
1. Introduction Microfluidic paper-based analytical devices (μPAD) is a novel microfluidic analysis technology platform invented by Whitesides team of Harvard University in 2007 (Martinez et al., 2007). Because of its advantages including low cost, easy to use, disposable, portable, rapid analysis and simultaneous detection of multi-components, its influence in academia and industry is soaring day by day (Huang et al., 2017; Li et al., 2018; Xu et al., 2018). Compared with silicon, glass (Lee et al., 2018), polymer and other materials as the substrate of biosensor, the main component of filter paper is cellulose which can easily immobilize biological macromolecules such as enzyme, protein and DNA, besides its biocompatibility is better than other substrates (Gao et al., 2018; Kong et al., 2018; Xiao et al., 2017). μPAD has been widely used in clinical diagnosis, food quality, environment monitoring and other fields (Wang
et al., 2012). ECL is a powerful technique for the detection of trace samples (Yang et al., 2018), which has been widely used in drug eval uation, environmental monitoring, disease diagnosis and other fields because of its low background, high sensitivity, low cost and so on (Jian et al., 2018). Meanwhile, ECL technique has been introduced into μPAD in recent years (Ma et al., 2017). Many environmental factors can disturb the signal output, which may lead to false positive or false negative (Wang et al., 2016). ECL luminophore/co-reactant may be incompatible with the target and result in cross-interference (Zhang and Ding, 2017). To avoid these drawbacks, the closed bipolar electrode is applied to the ECL system. The closed BPE (Liu et al., 2018)is physically divided two separate zone (reporting zone and sensing zone) to provide inde pendent space for eliminating the influence, and the only current flow path is through BPE (Feng et al., 2014; Ge et al., 2018). BPE has attracted
** Corresponding author. * Corresponding author. E-mail addresses: [email protected] (Y. Wang), [email protected] (S. Ge). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.bios.2019.111917 Received 30 October 2019; Received in revised form 20 November 2019; Accepted 22 November 2019 Available online 23 November 2019 0956-5663/© 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Fangfang Wang, Biosensors and Bioelectronics, https://doi.org/10.1016/j.bios.2019.111917
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significant attention owing to its extensive practical value and wide spread applications, such as electrochemical analysis, biosensor, multi component screening of electrocatalysts and so on (Baek et al., 2018; Wu et al., 2015; Xu et al., 2017; Zhang et al., 2017). Recently, ECL detection based on BPEs has attracted extensive attention because of their intrinsic merits, such as its ease of integration into portable devices and the lack of direct electrical connection required between it and an external power supply (Ge et al., 2014; Wang et al., 2017). As a traditional BPE substrate, indium tin oxide (ITO) is now widely applied to the field of BPE-ECL sensing platform (Shi et al., 2016; Wu et al., 2015). However, the ITO band is constructed by photolithography and wet chemical etching techniques, its process is complicated and time-consuming. What’s more, H2O2 at ITO is easily reduced under a high driving voltage, causing the damage of ITO. Paper possesses porous structure, hydrophilic performance and biocompatibility (Liu et al., 2016). It can store the reagents on the device and immobilize the biomolecule without an additional layer. Conductive carbon ink has the advantages of good stability, easy use and low cost, which can be used for ink-jet printing or screen printing to prepare BPE-ECL biosensors (Feng et al., 2014; Ge et al., 2018). For the sake of improving the conductivity and increasing the performance of BPE, AuNPs is exploited to fabricate the BPE of sensing platform (Zheng et al., 2018). To prevent the flow of fluid be tween the reporting cell and sensing cell, waterproof electronic bridge was prepared via printing wax between the cathode and the anode of BPE electrode. Ru(bpy)2þ 3 /TPrA system is a commonly-used ECL system in three-electrode system, which is less used in BPE system (Pang et al., 2005; Yang et al., 2018). The combination of Ru(bpy)2þ 3 /TPrA (Villani et al., 2018)system and BPE is a novel research topics in biosensor. With the aid of AuPd NPs mimetic enzyme and hybridization chain reaction (Bi et al., 2017; Liu et al., 2018), the development of multiple signal amplification would provide a new approach to further boost the bio sensing performance (Huang et al., 2018; Lv et al., 2018; Wang et al., 2018b). It is of great significance for this BPE system to simplify the research method and obtain higher sensitivity (Lu et al., 2018). BPE-ECL has a broad application prospect to satisfy the requirements of current biomedical applications. In this work, combined with the merits of μPAD and BPE system, a closed Au-BPE ECL analysis system for rapid and sensitive detection of miRNA-155 was constructed, which consisted of paper, carbon driving electrode, reporting cell, sensing cell and Au-BPE. As shown in Scheme 1, the hairpin probe H1 was immobilized on the cathode surface of BPE via a stable Au–S bond (Labib et al., 2016). In the presence of
miRNA-155, the hairpin structure of H1 was opened to hybridize with the target miRNA-155. Since the combination of H1 and H2 was more stable than that of H1 and miRNA-155, H2 replaced the target miRNA-155 (Li et al., 2019; Yue et al., 2017, 2019). The released miRNA-155 was recycled, and other hairpin H1 would be initiated. After the end of the cycle, a large number of exposed areas of H1–H2 system could be combined with S1–AuPd NPs (Qi et al., 2018). On the basis of the charge balance, AuPd NPs catalyzed the H2O2 reduction, the reduction rate of H2O2 at the cathode of BPE equaled to the oxidation rate of ECL luminophore and coreactant at the anode of BPE. Therefore, the target in the sensing cell could be quantitatively detected by measuring the ECL signal of the reporting cell. The new miRNA-155 detection method provides a promising platform for clinical detection of other biomolecules (Wang et al., 2018a). 2. Experimental section 2.1. Materials and apparatus The materials and apparatus are described in the Supplementary Material. 2.2. Preparation of AuPd NPs and S1– AuPd NPs AuPd NPs were synthesized by a facile wet-chemical reduction ac cording to the literature report with a slight modification. Briefly, Na2PdCl4 and HAuCl4 were used as metal sources, and ascorsbic acid (AA) as reducing agent in the presence of chlorohexadecyl pyridine. The details could be described as follows: first, 1.6 mL of 10 mM Na2PdCl4 aqueous solution and 164 μL of 1% HAuCl4 aqueous solution were evenly mixed with magnetic stirring. Then 72 mg of cetylpyridine chloride (HDPC) solid was dissolved in 20 mL of ultrapure water. After a while, 1.2 mL of 0.1 M AA solution that fresh prepared was added to the above mixed solution quickly under continuous stirring and the mixture was allowed to stand at 35 � C for 3 h. Finally, it was centrifuged and washed with ultrapure water for five times. After washing, the synthe sized AuPd NPs were stored at 4 � C in the refrigerator to use. The steps for the synthesis of functional S1– AuPd NPs can be sum marized as follows. The obtained AuPd NPs were dispersed by ultra sound for 2 h, and then S1 was added in the above solution stirring for 24 h at 4 � C. After that the mixed solution of S1 (2 μM) and AuPd NPs were placed on an oscillator for 12 h. The mixture of S1– AuPd NPs was
Scheme 1. Preparation of the ECL biosensor for the detection of miRNA-155 based on a paper-based BPE: the process of the cathode are the immobilization of H1, stand displacement reaction of H1, H2 and miRNA-155, the cycle of target, the immobilization of S1–AuPd and H2O2 is reduced; the process of the anode are the Ru (bpy)2þ 3 /TPrA system is oxidized and an ECL signal appears. 2
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prepared through the interaction between –NH2 groups of S1 and AuPd NPs, washed with ultrapure water to remove the unbound S1. The prepared S1– AuPd NPs was resuspended in 1 mL of ultrapure water at 4 � C for the subsequent experiment.
3. Results and discussion 3.1. Mechanism of the proposed biosensor The manufacturing process of BPE devices and the principle of electrochemiluminescence strategy for miRNA-155 detection were shown in Schemes 1. As a result of the connection between cathode and anode, the electron transfer process in BPE was electrically coupled with ECL reaction. The electron transfer process took place between the two poles of BPE. In detail, a DC power supply was connected across the bipolar electrode and a suitable driving voltage was carried out, folded the paper chip so that the BPE was in full contact with the sensing cell and the reporting cell. At this time, in the cathode region of the BPE, H2O2 in the sensing unit underwent a reduction reaction under the catalysis of AuPd NPs, which was a process of obtaining electrons. At the same time, the Ru(bpy)2þ 3 /TPrA system in the reporting unit lost its electrons, which produced output light signal in the anode region of the BPE. Therefore, with the rules of charge balance, the poles made the light output of the anode showed a quantitative relationship with the electrochemical reduction process at the cathode. Two hairpin DNA fragments (H1 and H2) was used to constructed the biosensor. In the absence of a target, they could maintain a stable structure and couldn’t hybridize automatically. Firstly, H1 bound to the cathode of BPE via the Au–S bond, and MCH was used to block nonspecific binding sites. After the target was added, the stem ring structure of H1 was opened, crossed with target to form hybrid double-stranded DNA. Because the H1–H2 double-stranded structure was more stable than the H1-target hybrid complex, when hybridized with H1, H2 took the place of the target and released it. After the target molecule was released, it continually participated in the next H2 cycle. The base of H2 was not complete complementary with H1, the reminder fragment of H2 was comple mentary with the S1 fragment, which the other end of S1 was labeled AuPd NPs. H2O2 was reduced under the catalysis of AuPd NPs. In addition, due to the co-reactant of TPrA, the reaction rate and intensity of ECL have been greatly improved. Therefore, using AuPd NPs as catalyst and TPrA as co-reactant could improve the detection sensitivity of miRNA electrochemiluminescence biosensor. The system of BPE biosensor was divided to two isolated area including sensing cell and reporting cell, which enhanced the selectivity. The system of BPE was more fascinating than the traditional three-electrode system.
2.3. Fabrication of the bipolar electrode The paper-based BPE-ECL sensor (4 � 4 cm) was composed of two hydrophilic regions (blank reaction area 10 � 10 mm), bipolar electrode printing area (5 � 15 mm), hydrophobic area (hydrophobic printing area) and water proof salt bridge. As shown in Fig. S1, the paper-based BPE chip was designed by AI software. The wax pattern was printed with a printer. Next, the waxed paper was put in the oven (130 � C, 1 min), whereafter, the wax melt and penetrated into the paper before taken out. The printed wax zone is hydrophobic zone, and nonprinted wax zone is hydrophilic zone. The driving electrodes were consisted of two carbon electrodes, which was printed by screen printing techniques. According to our previous work, it provided an easy way to prepare the Au-BPE, in which the AuNPs were seeded on the cellulose fibers through the in-situ growth of AuNPs layer on the surfaces of cellulose fibers in the BPE to enhance the conductivity of the BPE (Zheng et al., 2018). To prevent the fluid flow between the reporting cell and sensing cell, waterproof elec tronic bridge was prepared via the second wax-printed on the middle part of the BPE electrode, which allowed the electronic transfer via the bipolar and prevented the fluid exchange between reporting cell and sensing cell. The prepared paper-based chip was folded in half along the centerline, and the two cells were in full contact with the BPE on the other side as an electronic conductor (see Supplementary Material Fig. S2 for the picture of paper-based closed bipolar electrode). 2.4. Preparation of the MiRNA-155 electrochemiluminescence biosensor All the DNA were heated (95 � C), annealed (2 min) and cooled to room temperature. Firstly, 10 μL H1 (2 μM) was added to the cathode of Au-BPE and allowed to stand for 16 h (room temperature). H1 was anchored on the cathode via the S–Au bond. Next, the unloaded H1 was washed off with ultrapure water. After the surface was dried, the nonspecific binding site was blocked by MCH for 30 min to avoid the nonspecific adsorption. Then the uncombined MCH was rinsed off. The cathode immobilized H1 was further modified by a mixed solution of 10 μL H2 (2 μM) and miRNA-155 with concentration gradient, and then incubated at 37 � C (2 h) before the unloaded chain was washed. Next, S1–AuPd NPs (10 μL) was added to the above cathode area and culti vated at 4 � C for 2 h. Then the modified electrode was cleaned, dried and stored (4 � C). The ECL signal could be measured and provided the cor responding quantitative standard for the detection of miRNA.
3.2. Characterization of Au-BPE and AuPd NPs In the BPE-ECL process, the electrode materials has a very important influence on the efficiency of the overall sensing system (Fiorani et al., 2018). Several different electrode materials were tested and compared, and AuNPs was chosen to modify the cellulose in the bipolar electrode region. Fig. 1A and Fig. 1B show the SEM images of the bare paper and paper fibers loaded with Au NPs, respectively. As shown in Fig. 1C, the thin Au NPs uniform load on the surface of the paper fiber, which is conducive to the stable load of H1 on the sensing device through the Au–S bond. Porous bimetallic SEM image (Fig. 1D) and transmission electron microscopy (TEM) (Fig. 1E) show the structure of AuPd NPs. It shows that AuPd NPs like-spherical morphology are a solid core with the shell of multi-branched nanostructure. The characteristic of SEM and TEM are identical in shape and size, each nanostructure is porous, with highly branched subunits ranging in size about 60 nm. The formation of AuPd NPs was further confirmed by the analysis of Au4f and Pd3d by X-ray photoelectron spectroscopy (XPS) (Fig. 1F and Fig. 1G). AuPd NPs were characterized with XRD (Fig. 1H), indicating the successful prep aration of AuPd NPs. The element types and contents of AuPd NPs were verified by energy dispersive X-ray spectroscopy (EDS) (Fig. 1I). The results show that the interface was composed of Au and Pd elements, indicating the formation of AuPd NPs composite. Thus, all these data demonstrated the formation of Au-BPE and bimetallic porous AuPd NPs was successfully prepared.
2.5. ECL measurements The operation procedure of paper-based BPE-ECL detected miRNA is as follows. The prepared disposable paper chip was placed on the sub strate and the required solution was dripped on the sensing cell (0.1 M PBS (pH 7.0) and 0.5 nM H2O2) and the reporting cell (0.1 M PBS (pH 7.0), 1 mM Ru(bpy)2þ 3 and 50 mM TPrA), respectively. The paper chip was folded in half along the centerline so that the Au-BPE was fully integrated with the sensing cell and the reporting cell. The positive and negative electrodes on the direct current (DC) power supply were con nected to the graphite drive electrode of the paper chip. And put it in a cassette, the paper-based BPE was adjusted to the appropriate position so that it faced the photomultiplier tube. Close the cassette so that it was completely sheltered from light. The DC power supply with a suitable driving voltage was turned on and the ECL signal appeared on the display screen. 3
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Fig. 1. (A) SEM image of bare paper. (B) and (C) SEM image of AuNPs seeded on the surface of the cellulose. (D) SEM image, (E) TEM image, (F) and (G) XPS analysis, (H) XRD and (I) EDS analysis of AuPd NPs.
3.3. Electrochemiluminescence characterization of the BPE-ECL biosensor
method. The successful preparation of electrochemiluminescence biosensor can be proved by using CV curve. The step-by-step modified electrode was implemented in the buffer containing PBS (pH 7.0, 0.1 M) and 5.0 mM [Fe(CN)6]3-/4- for CV scanning. As shown in Fig. 2A, the
Cyclic voltammetry (CV) (Shao et al., 2018) is not only used as a quantitative analysis method, but also as an electrochemical research
Fig. 2. (A) CV and (B) EIS of electrodes with different modified in PBS (0.1 M pH 7.0) including 5.0 mM [Fe(CN)6]3-/4- (a) unmodified bare BPE, (b) Au-BPE, (c) H1/ Au-BPE, (d) MCH/H1/Au-BPE, (e) H2/miRNA-155/MCH/H1/Au-BPE, (f) S1–AuPd NPs/H2/MCH/H1/Au-BPE. 4
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curves. The linear range of concentration was 1.0 � 10 12–1.0 � 10 5 M, and the linear regression equation was I ¼ 17018 þ 1222 lgc with the regression coefficient (R2) was 0.9969. The detection limit was calcu lated to be 0.67 pM (S/N ¼ 3). The low detection limit was because of the large loading of Ru(bpy)2þ 3 /TPrA and the catalytic effect of AuPd NPs on the reduction of H2O2. (Performance of miRNA detection con trasted with other works see Supplementary Material Table S1).
bare paper did not exhibit one well-defined redox peaks (Fig. 2A, curve a). When AuNPs was loaded on bare paper to prepare the BPE, the CV peak current sharply increased, the CV curves showed that the peak current of AuNPs modified electrode increased significantly (Fig. 2A, curve b), ascribing to AuNPs had a large effective surface and good conductivity. When H1 was dropped into the Au-BPE, the redox peak current decreased (Fig. 2A, curve c), indicating that H1 reduced electron transfer. After the MCH blocked specific site, the peak current obviously decreased (Fig. 2A, curve d). After adding the mixed solution of H2 and miRNA-155, the conductivity of the electrode decreased and the peak current further decreased (Fig. 2A, curve e). Finally, after the modifi cation of S1–AuPd NPs on the modified electrode, due to the increase of steric resistance, hindered the diffusion of [Fe(CN)6]3-/4- toward the surfaces of BPE, the redox peak current further decreased (Fig. 2A, curve f). Therefore, the successful assembly of biosensors could be proved by the reasonable change of CV peak current. Electrochemical impedance spectroscopy (EIS) (Ge et al., 2017; Zhang et al., 2018) is used to detect the interfacial properties of the modified electrode. The modified electrode was measured in a buffer solution containing [Fe(CN)6]3-/4-. In the high frequency region, bare BPE showed a semicircle (Fig. 2B, curved b). The conductivity of AuNPs modified BPE increased greatly and the charge transfer resistance (Ret) decreased obviously (Fig. 2B, curve a). When H1 was anchored to the Au-BPE, because the DNA strand was a non-conductive substance, it could be seen that the Ret increased (Fig. 2B, curve c). After the specific sites of BPE cathode were blocked by MCH, Ret increased continuously (Fig. 2B, curve d). Then, the mixed solution of H2 and miRNA-155 was added, the Ret further increased (Fig. 2B, curve e). Finally, when the S1–AuPd NPs was connected to H2, the steric hindrance was further increased and the Ret reached the maximum (Fig. 2B, curve f).
3.6. Selectivity, stability and reproducibility of the ECL biosensor For purpose of studying the specificity of the constructed BPE-ECL biosensor, the effect of a variety of miRNA (including let-7a, miRNA210, sRNA, nRNA, and miRNA-155) on paper-based BPE-ECL sensing platform were detected. It could be seen from Fig. 5A that the strongest ECL intensity was observed in the presence of miRNA-155. The ECL intensity of other RNA was obviously lower than that of target, which proved that ECL biosensor had acceptable specificity. The stability of the BPE-ECL biosensor was evaluated by consecutive continuous cyclic potential scanning for the same concentrations of miRNA-155. As shown in Fig. 5B, a stable ECL intensity were obtained. The prepared biosensor was stored in a refrigerator at 4 � C. The stability of biosensor was 98.1% in the first five days and then decreased by 3.6% in the next 10 days. It showed no evidently descend in ECL intensity after 15 days, it retained 95.5% of its initial response. The results indicated that the prepared biosensor had favourable stability for the detection of miRNA-155. The reproducibility of the BPE-ECL biosensor was assessed by mean of the differences (relative standard deviation (RSD)) of the intra-assays and inter-assays. Here we used standard solution including three different concentrations of miRNA (10 pM、10 nM、10 μM) to evaluate the RSD (n ¼ 5). The experimental data indicated that the RSD values of the intra-assay were 4.6%, 5.8% and 3.7% at 10 pM, 10 nM and 10 μM miRNA-155, whereas the RSD values of the inter-assay using different batches of the BPE-ECL biosensor were 8.9%, 7.3% and 9.1% at 10 pM, 10 nM and 10 μM miRNA-155, respectively. Thus, the reproducibility of the proposed biosensor was satisfied.
3.4. Optimization of experimental condition Because the conductivity of the bipolar electrode affects the detec tion sensitivity. As shown in Fig. 3A, it was obvious that the complete Au-BPE was the best choice for this BPE-ECL biosensor. (Detailed description of Fig. 3A and Fig. 3B see the Supplementary Material).
4. Conclusion
3.5. Detection of MiRNA-155
In summary, the paper-based Au closed BPE ECL biosensor system was successfully constructed. This method has many advantages: first, Au closed BPE provided the excellent conductivity than the graphite electrode. Second, the closed bipolar electrode system is a good choice compared with the three-electrode system, because it can distinguish the anode and cathode into two separate compartments, avoid any inter ference and have higher current efficiency. Third, AuPd NPs as a catalyst
Fig. 4 is a schematic diagram of the detection of miRNA-155 by the ECL method. Under the optimum conditions (see Fig. 3 and Supple mentary Material Fig. S3), the concentration of miRNA-155 could be reflected by ECL intensity. As shown in Fig. 4B, the logarithm of the concentration of miRNA-155 was proportional to the ECL intensity. Here different concentrations of miRNA-155 were selected to make standard
Fig. 3. (A) Optimization of electrical conductivity of BPE (a) Au NPs were all grown in the BPE region, (b) the BPE area is screen-printed with graphite electrodes loaded with gold at both ends, (c) screen printed graphite electrode in BPE area. (B) influence of the concentration of Ru(bpy)2þ 3 . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 5
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Fig. 4. (A) ECL responses of the biosensor with different concentrations of miRNA-155: 1 mM, 0.1 mM, 0.01 mM, 1 μM, 0.1 μM, 0.01 μM, 1 nM, 0.1 nM, 0.01 nM, 1 pM; under the condition of PBS (0.1 M, pH 7.0) including 0.5 nM H2O2. (B) The curve of ECL intensity vs the logarithm of miRNA-155 concentration.
Fig. 5. (A) The specificity of this proposed BPE-ECL biosensor for different interfering substance: (a) blank solution (0 fM miRNA-155), (b) let-7a (2 μM), (c) miRNA210 (2 μM), (d) sRNA (2 μM), (e) nRNA (2 μM), (f) miRNA-155 (2 μM). (B) The stability for different storage conditions of the proposed BPE-ECL biosensor (a) in a freezer, (b) in a refrigerator and (c) at room temperature.
could catalyze the reduction of H2O2 for signal amplification. Moreover, compared with other methods, the Au closed bipolar electrode electro chemiluminescence biosensor has good sensitivity and selectivity to miRNA-155. Therefore, this principle not only greatly expands the application of high performance BPE, but also provides a novel avenue for the detection of miRNA in clinical application, which can be applied other analytes.
items of University” of Jinan (2018GXRC001); the Taishan Scholars program and Case-by-Case Project for Top Outstanding Talents of Jinan. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.bios.2019.111917.
Declaration of competing interest
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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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CRediT authorship contribution statement Fangfang Wang: Conceptualization, Methodology, Writing - orig inal draft. Cuiping Fu: Investigation, Formal analysis. Chuan Huang: Visualization, Data curation. Na Li: Resources, Visualization. Yanhu Wang: Writing - review & editing, Resources. Shenguang Ge: Conceptualization, Supervision, Funding acquisition. Jinghua Yu: Funding acquisition, Project administration. Acknowledgements This work was supported by the National Natural Science Foundation of China (21775055, 21575051, 21874055); Excellent Youth Innovation Team in Universities of Shandong (2019KJC016); The Project of “20 6
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Biosensors and Bioelectronics journal homepage: http://www.elsevier.com/locate/bios
Paper-based closed Au-Bipolar electrode electrochemiluminescence sensing platform for the detection of miRNA-155 Fangfang Wang a, 1, Cuiping Fu a, 1, Chuan Huang a, 1, Na Li b, Yanhu Wang c, **, Shenguang Ge a, *, Jinghua Yu b a
Collaborative Innovation Center of Technology and Equipment for Biological Diagnosis and Therapy in Universities of Shandong, Institute for Advanced Interdisciplinary Research, University of Jinan, Jinan, 250022, PR China School of Chemistry and Chemical Engineering, University of Jinan, Jinan, 250022, PR China c Shandong Key Laboratory of TCM Quality Control Technology, Shandong Analysis and Test Center, Qilu University of Technology (Shandong Academy of Sciences), Jinan, 250014, PR China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Electrochemiluminescence Bipolar electrode MicroRNA-155 AuPd nanoparticles.
This paper introduces a paper-based closed Au-bipolar electrode (BPE) biosensing system for the rapid and sensitive electrochemiluminescence (ECL) detection of miRNA-155. This microfluidic paper-based sensing platform is formed by wax-printing technology, screen printing method and in-situ Au nanoparticles (NPs) growth to form hydrophilic cells, hydrophobic boundaries, water proof electronic bridge, driving electrode re gions and bipolar electrode regions. For rapid and sensitive detection, the cathode of bipolar electrode was modified with the prepared DNA (S1)–AuPd NPs by hybridization chain reaction, in which the target could initiate multiple cycles reaction to load more AuPd NPs which catalyzed H2O2 reduction. In addition, a classical ECL system tris (2,20 -bipyridine) ruthenium (II)- tripropylamine (Ru(bpy)2þ 3 /TPrA) exists at the anode of the bipolar electrode. Due to the charge balance between the anode and the cathode of BPE, the ECL signal response of Ru(bpy)2þ 3 /TPrA system was enhanced in the reporting cell. The intensity of ECL was quantitatively correlated with the concentration of miRNA-155 in the range of 1 pM–10 μM with the detection limit 0.67 pM. Moreover, this method paves a novel way for highly sensitive detection of miRNA-155 in clinical application.
1. Introduction Microfluidic paper-based analytical devices (μPAD) is a novel microfluidic analysis technology platform invented by Whitesides team of Harvard University in 2007 (Martinez et al., 2007). Because of its advantages including low cost, easy to use, disposable, portable, rapid analysis and simultaneous detection of multi-components, its influence in academia and industry is soaring day by day (Huang et al., 2017; Li et al., 2018; Xu et al., 2018). Compared with silicon, glass (Lee et al., 2018), polymer and other materials as the substrate of biosensor, the main component of filter paper is cellulose which can easily immobilize biological macromolecules such as enzyme, protein and DNA, besides its biocompatibility is better than other substrates (Gao et al., 2018; Kong et al., 2018; Xiao et al., 2017). μPAD has been widely used in clinical diagnosis, food quality, environment monitoring and other fields (Wang
et al., 2012). ECL is a powerful technique for the detection of trace samples (Yang et al., 2018), which has been widely used in drug eval uation, environmental monitoring, disease diagnosis and other fields because of its low background, high sensitivity, low cost and so on (Jian et al., 2018). Meanwhile, ECL technique has been introduced into μPAD in recent years (Ma et al., 2017). Many environmental factors can disturb the signal output, which may lead to false positive or false negative (Wang et al., 2016). ECL luminophore/co-reactant may be incompatible with the target and result in cross-interference (Zhang and Ding, 2017). To avoid these drawbacks, the closed bipolar electrode is applied to the ECL system. The closed BPE (Liu et al., 2018)is physically divided two separate zone (reporting zone and sensing zone) to provide inde pendent space for eliminating the influence, and the only current flow path is through BPE (Feng et al., 2014; Ge et al., 2018). BPE has attracted
** Corresponding author. * Corresponding author. E-mail addresses: [email protected] (Y. Wang), [email protected] (S. Ge). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.bios.2019.111917 Received 30 October 2019; Received in revised form 20 November 2019; Accepted 22 November 2019 Available online 23 November 2019 0956-5663/© 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Fangfang Wang, Biosensors and Bioelectronics, https://doi.org/10.1016/j.bios.2019.111917
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significant attention owing to its extensive practical value and wide spread applications, such as electrochemical analysis, biosensor, multi component screening of electrocatalysts and so on (Baek et al., 2018; Wu et al., 2015; Xu et al., 2017; Zhang et al., 2017). Recently, ECL detection based on BPEs has attracted extensive attention because of their intrinsic merits, such as its ease of integration into portable devices and the lack of direct electrical connection required between it and an external power supply (Ge et al., 2014; Wang et al., 2017). As a traditional BPE substrate, indium tin oxide (ITO) is now widely applied to the field of BPE-ECL sensing platform (Shi et al., 2016; Wu et al., 2015). However, the ITO band is constructed by photolithography and wet chemical etching techniques, its process is complicated and time-consuming. What’s more, H2O2 at ITO is easily reduced under a high driving voltage, causing the damage of ITO. Paper possesses porous structure, hydrophilic performance and biocompatibility (Liu et al., 2016). It can store the reagents on the device and immobilize the biomolecule without an additional layer. Conductive carbon ink has the advantages of good stability, easy use and low cost, which can be used for ink-jet printing or screen printing to prepare BPE-ECL biosensors (Feng et al., 2014; Ge et al., 2018). For the sake of improving the conductivity and increasing the performance of BPE, AuNPs is exploited to fabricate the BPE of sensing platform (Zheng et al., 2018). To prevent the flow of fluid be tween the reporting cell and sensing cell, waterproof electronic bridge was prepared via printing wax between the cathode and the anode of BPE electrode. Ru(bpy)2þ 3 /TPrA system is a commonly-used ECL system in three-electrode system, which is less used in BPE system (Pang et al., 2005; Yang et al., 2018). The combination of Ru(bpy)2þ 3 /TPrA (Villani et al., 2018)system and BPE is a novel research topics in biosensor. With the aid of AuPd NPs mimetic enzyme and hybridization chain reaction (Bi et al., 2017; Liu et al., 2018), the development of multiple signal amplification would provide a new approach to further boost the bio sensing performance (Huang et al., 2018; Lv et al., 2018; Wang et al., 2018b). It is of great significance for this BPE system to simplify the research method and obtain higher sensitivity (Lu et al., 2018). BPE-ECL has a broad application prospect to satisfy the requirements of current biomedical applications. In this work, combined with the merits of μPAD and BPE system, a closed Au-BPE ECL analysis system for rapid and sensitive detection of miRNA-155 was constructed, which consisted of paper, carbon driving electrode, reporting cell, sensing cell and Au-BPE. As shown in Scheme 1, the hairpin probe H1 was immobilized on the cathode surface of BPE via a stable Au–S bond (Labib et al., 2016). In the presence of
miRNA-155, the hairpin structure of H1 was opened to hybridize with the target miRNA-155. Since the combination of H1 and H2 was more stable than that of H1 and miRNA-155, H2 replaced the target miRNA-155 (Li et al., 2019; Yue et al., 2017, 2019). The released miRNA-155 was recycled, and other hairpin H1 would be initiated. After the end of the cycle, a large number of exposed areas of H1–H2 system could be combined with S1–AuPd NPs (Qi et al., 2018). On the basis of the charge balance, AuPd NPs catalyzed the H2O2 reduction, the reduction rate of H2O2 at the cathode of BPE equaled to the oxidation rate of ECL luminophore and coreactant at the anode of BPE. Therefore, the target in the sensing cell could be quantitatively detected by measuring the ECL signal of the reporting cell. The new miRNA-155 detection method provides a promising platform for clinical detection of other biomolecules (Wang et al., 2018a). 2. Experimental section 2.1. Materials and apparatus The materials and apparatus are described in the Supplementary Material. 2.2. Preparation of AuPd NPs and S1– AuPd NPs AuPd NPs were synthesized by a facile wet-chemical reduction ac cording to the literature report with a slight modification. Briefly, Na2PdCl4 and HAuCl4 were used as metal sources, and ascorsbic acid (AA) as reducing agent in the presence of chlorohexadecyl pyridine. The details could be described as follows: first, 1.6 mL of 10 mM Na2PdCl4 aqueous solution and 164 μL of 1% HAuCl4 aqueous solution were evenly mixed with magnetic stirring. Then 72 mg of cetylpyridine chloride (HDPC) solid was dissolved in 20 mL of ultrapure water. After a while, 1.2 mL of 0.1 M AA solution that fresh prepared was added to the above mixed solution quickly under continuous stirring and the mixture was allowed to stand at 35 � C for 3 h. Finally, it was centrifuged and washed with ultrapure water for five times. After washing, the synthe sized AuPd NPs were stored at 4 � C in the refrigerator to use. The steps for the synthesis of functional S1– AuPd NPs can be sum marized as follows. The obtained AuPd NPs were dispersed by ultra sound for 2 h, and then S1 was added in the above solution stirring for 24 h at 4 � C. After that the mixed solution of S1 (2 μM) and AuPd NPs were placed on an oscillator for 12 h. The mixture of S1– AuPd NPs was
Scheme 1. Preparation of the ECL biosensor for the detection of miRNA-155 based on a paper-based BPE: the process of the cathode are the immobilization of H1, stand displacement reaction of H1, H2 and miRNA-155, the cycle of target, the immobilization of S1–AuPd and H2O2 is reduced; the process of the anode are the Ru (bpy)2þ 3 /TPrA system is oxidized and an ECL signal appears. 2
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prepared through the interaction between –NH2 groups of S1 and AuPd NPs, washed with ultrapure water to remove the unbound S1. The prepared S1– AuPd NPs was resuspended in 1 mL of ultrapure water at 4 � C for the subsequent experiment.
3. Results and discussion 3.1. Mechanism of the proposed biosensor The manufacturing process of BPE devices and the principle of electrochemiluminescence strategy for miRNA-155 detection were shown in Schemes 1. As a result of the connection between cathode and anode, the electron transfer process in BPE was electrically coupled with ECL reaction. The electron transfer process took place between the two poles of BPE. In detail, a DC power supply was connected across the bipolar electrode and a suitable driving voltage was carried out, folded the paper chip so that the BPE was in full contact with the sensing cell and the reporting cell. At this time, in the cathode region of the BPE, H2O2 in the sensing unit underwent a reduction reaction under the catalysis of AuPd NPs, which was a process of obtaining electrons. At the same time, the Ru(bpy)2þ 3 /TPrA system in the reporting unit lost its electrons, which produced output light signal in the anode region of the BPE. Therefore, with the rules of charge balance, the poles made the light output of the anode showed a quantitative relationship with the electrochemical reduction process at the cathode. Two hairpin DNA fragments (H1 and H2) was used to constructed the biosensor. In the absence of a target, they could maintain a stable structure and couldn’t hybridize automatically. Firstly, H1 bound to the cathode of BPE via the Au–S bond, and MCH was used to block nonspecific binding sites. After the target was added, the stem ring structure of H1 was opened, crossed with target to form hybrid double-stranded DNA. Because the H1–H2 double-stranded structure was more stable than the H1-target hybrid complex, when hybridized with H1, H2 took the place of the target and released it. After the target molecule was released, it continually participated in the next H2 cycle. The base of H2 was not complete complementary with H1, the reminder fragment of H2 was comple mentary with the S1 fragment, which the other end of S1 was labeled AuPd NPs. H2O2 was reduced under the catalysis of AuPd NPs. In addition, due to the co-reactant of TPrA, the reaction rate and intensity of ECL have been greatly improved. Therefore, using AuPd NPs as catalyst and TPrA as co-reactant could improve the detection sensitivity of miRNA electrochemiluminescence biosensor. The system of BPE biosensor was divided to two isolated area including sensing cell and reporting cell, which enhanced the selectivity. The system of BPE was more fascinating than the traditional three-electrode system.
2.3. Fabrication of the bipolar electrode The paper-based BPE-ECL sensor (4 � 4 cm) was composed of two hydrophilic regions (blank reaction area 10 � 10 mm), bipolar electrode printing area (5 � 15 mm), hydrophobic area (hydrophobic printing area) and water proof salt bridge. As shown in Fig. S1, the paper-based BPE chip was designed by AI software. The wax pattern was printed with a printer. Next, the waxed paper was put in the oven (130 � C, 1 min), whereafter, the wax melt and penetrated into the paper before taken out. The printed wax zone is hydrophobic zone, and nonprinted wax zone is hydrophilic zone. The driving electrodes were consisted of two carbon electrodes, which was printed by screen printing techniques. According to our previous work, it provided an easy way to prepare the Au-BPE, in which the AuNPs were seeded on the cellulose fibers through the in-situ growth of AuNPs layer on the surfaces of cellulose fibers in the BPE to enhance the conductivity of the BPE (Zheng et al., 2018). To prevent the fluid flow between the reporting cell and sensing cell, waterproof elec tronic bridge was prepared via the second wax-printed on the middle part of the BPE electrode, which allowed the electronic transfer via the bipolar and prevented the fluid exchange between reporting cell and sensing cell. The prepared paper-based chip was folded in half along the centerline, and the two cells were in full contact with the BPE on the other side as an electronic conductor (see Supplementary Material Fig. S2 for the picture of paper-based closed bipolar electrode). 2.4. Preparation of the MiRNA-155 electrochemiluminescence biosensor All the DNA were heated (95 � C), annealed (2 min) and cooled to room temperature. Firstly, 10 μL H1 (2 μM) was added to the cathode of Au-BPE and allowed to stand for 16 h (room temperature). H1 was anchored on the cathode via the S–Au bond. Next, the unloaded H1 was washed off with ultrapure water. After the surface was dried, the nonspecific binding site was blocked by MCH for 30 min to avoid the nonspecific adsorption. Then the uncombined MCH was rinsed off. The cathode immobilized H1 was further modified by a mixed solution of 10 μL H2 (2 μM) and miRNA-155 with concentration gradient, and then incubated at 37 � C (2 h) before the unloaded chain was washed. Next, S1–AuPd NPs (10 μL) was added to the above cathode area and culti vated at 4 � C for 2 h. Then the modified electrode was cleaned, dried and stored (4 � C). The ECL signal could be measured and provided the cor responding quantitative standard for the detection of miRNA.
3.2. Characterization of Au-BPE and AuPd NPs In the BPE-ECL process, the electrode materials has a very important influence on the efficiency of the overall sensing system (Fiorani et al., 2018). Several different electrode materials were tested and compared, and AuNPs was chosen to modify the cellulose in the bipolar electrode region. Fig. 1A and Fig. 1B show the SEM images of the bare paper and paper fibers loaded with Au NPs, respectively. As shown in Fig. 1C, the thin Au NPs uniform load on the surface of the paper fiber, which is conducive to the stable load of H1 on the sensing device through the Au–S bond. Porous bimetallic SEM image (Fig. 1D) and transmission electron microscopy (TEM) (Fig. 1E) show the structure of AuPd NPs. It shows that AuPd NPs like-spherical morphology are a solid core with the shell of multi-branched nanostructure. The characteristic of SEM and TEM are identical in shape and size, each nanostructure is porous, with highly branched subunits ranging in size about 60 nm. The formation of AuPd NPs was further confirmed by the analysis of Au4f and Pd3d by X-ray photoelectron spectroscopy (XPS) (Fig. 1F and Fig. 1G). AuPd NPs were characterized with XRD (Fig. 1H), indicating the successful prep aration of AuPd NPs. The element types and contents of AuPd NPs were verified by energy dispersive X-ray spectroscopy (EDS) (Fig. 1I). The results show that the interface was composed of Au and Pd elements, indicating the formation of AuPd NPs composite. Thus, all these data demonstrated the formation of Au-BPE and bimetallic porous AuPd NPs was successfully prepared.
2.5. ECL measurements The operation procedure of paper-based BPE-ECL detected miRNA is as follows. The prepared disposable paper chip was placed on the sub strate and the required solution was dripped on the sensing cell (0.1 M PBS (pH 7.0) and 0.5 nM H2O2) and the reporting cell (0.1 M PBS (pH 7.0), 1 mM Ru(bpy)2þ 3 and 50 mM TPrA), respectively. The paper chip was folded in half along the centerline so that the Au-BPE was fully integrated with the sensing cell and the reporting cell. The positive and negative electrodes on the direct current (DC) power supply were con nected to the graphite drive electrode of the paper chip. And put it in a cassette, the paper-based BPE was adjusted to the appropriate position so that it faced the photomultiplier tube. Close the cassette so that it was completely sheltered from light. The DC power supply with a suitable driving voltage was turned on and the ECL signal appeared on the display screen. 3
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Fig. 1. (A) SEM image of bare paper. (B) and (C) SEM image of AuNPs seeded on the surface of the cellulose. (D) SEM image, (E) TEM image, (F) and (G) XPS analysis, (H) XRD and (I) EDS analysis of AuPd NPs.
3.3. Electrochemiluminescence characterization of the BPE-ECL biosensor
method. The successful preparation of electrochemiluminescence biosensor can be proved by using CV curve. The step-by-step modified electrode was implemented in the buffer containing PBS (pH 7.0, 0.1 M) and 5.0 mM [Fe(CN)6]3-/4- for CV scanning. As shown in Fig. 2A, the
Cyclic voltammetry (CV) (Shao et al., 2018) is not only used as a quantitative analysis method, but also as an electrochemical research
Fig. 2. (A) CV and (B) EIS of electrodes with different modified in PBS (0.1 M pH 7.0) including 5.0 mM [Fe(CN)6]3-/4- (a) unmodified bare BPE, (b) Au-BPE, (c) H1/ Au-BPE, (d) MCH/H1/Au-BPE, (e) H2/miRNA-155/MCH/H1/Au-BPE, (f) S1–AuPd NPs/H2/MCH/H1/Au-BPE. 4
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curves. The linear range of concentration was 1.0 � 10 12–1.0 � 10 5 M, and the linear regression equation was I ¼ 17018 þ 1222 lgc with the regression coefficient (R2) was 0.9969. The detection limit was calcu lated to be 0.67 pM (S/N ¼ 3). The low detection limit was because of the large loading of Ru(bpy)2þ 3 /TPrA and the catalytic effect of AuPd NPs on the reduction of H2O2. (Performance of miRNA detection con trasted with other works see Supplementary Material Table S1).
bare paper did not exhibit one well-defined redox peaks (Fig. 2A, curve a). When AuNPs was loaded on bare paper to prepare the BPE, the CV peak current sharply increased, the CV curves showed that the peak current of AuNPs modified electrode increased significantly (Fig. 2A, curve b), ascribing to AuNPs had a large effective surface and good conductivity. When H1 was dropped into the Au-BPE, the redox peak current decreased (Fig. 2A, curve c), indicating that H1 reduced electron transfer. After the MCH blocked specific site, the peak current obviously decreased (Fig. 2A, curve d). After adding the mixed solution of H2 and miRNA-155, the conductivity of the electrode decreased and the peak current further decreased (Fig. 2A, curve e). Finally, after the modifi cation of S1–AuPd NPs on the modified electrode, due to the increase of steric resistance, hindered the diffusion of [Fe(CN)6]3-/4- toward the surfaces of BPE, the redox peak current further decreased (Fig. 2A, curve f). Therefore, the successful assembly of biosensors could be proved by the reasonable change of CV peak current. Electrochemical impedance spectroscopy (EIS) (Ge et al., 2017; Zhang et al., 2018) is used to detect the interfacial properties of the modified electrode. The modified electrode was measured in a buffer solution containing [Fe(CN)6]3-/4-. In the high frequency region, bare BPE showed a semicircle (Fig. 2B, curved b). The conductivity of AuNPs modified BPE increased greatly and the charge transfer resistance (Ret) decreased obviously (Fig. 2B, curve a). When H1 was anchored to the Au-BPE, because the DNA strand was a non-conductive substance, it could be seen that the Ret increased (Fig. 2B, curve c). After the specific sites of BPE cathode were blocked by MCH, Ret increased continuously (Fig. 2B, curve d). Then, the mixed solution of H2 and miRNA-155 was added, the Ret further increased (Fig. 2B, curve e). Finally, when the S1–AuPd NPs was connected to H2, the steric hindrance was further increased and the Ret reached the maximum (Fig. 2B, curve f).
3.6. Selectivity, stability and reproducibility of the ECL biosensor For purpose of studying the specificity of the constructed BPE-ECL biosensor, the effect of a variety of miRNA (including let-7a, miRNA210, sRNA, nRNA, and miRNA-155) on paper-based BPE-ECL sensing platform were detected. It could be seen from Fig. 5A that the strongest ECL intensity was observed in the presence of miRNA-155. The ECL intensity of other RNA was obviously lower than that of target, which proved that ECL biosensor had acceptable specificity. The stability of the BPE-ECL biosensor was evaluated by consecutive continuous cyclic potential scanning for the same concentrations of miRNA-155. As shown in Fig. 5B, a stable ECL intensity were obtained. The prepared biosensor was stored in a refrigerator at 4 � C. The stability of biosensor was 98.1% in the first five days and then decreased by 3.6% in the next 10 days. It showed no evidently descend in ECL intensity after 15 days, it retained 95.5% of its initial response. The results indicated that the prepared biosensor had favourable stability for the detection of miRNA-155. The reproducibility of the BPE-ECL biosensor was assessed by mean of the differences (relative standard deviation (RSD)) of the intra-assays and inter-assays. Here we used standard solution including three different concentrations of miRNA (10 pM、10 nM、10 μM) to evaluate the RSD (n ¼ 5). The experimental data indicated that the RSD values of the intra-assay were 4.6%, 5.8% and 3.7% at 10 pM, 10 nM and 10 μM miRNA-155, whereas the RSD values of the inter-assay using different batches of the BPE-ECL biosensor were 8.9%, 7.3% and 9.1% at 10 pM, 10 nM and 10 μM miRNA-155, respectively. Thus, the reproducibility of the proposed biosensor was satisfied.
3.4. Optimization of experimental condition Because the conductivity of the bipolar electrode affects the detec tion sensitivity. As shown in Fig. 3A, it was obvious that the complete Au-BPE was the best choice for this BPE-ECL biosensor. (Detailed description of Fig. 3A and Fig. 3B see the Supplementary Material).
4. Conclusion
3.5. Detection of MiRNA-155
In summary, the paper-based Au closed BPE ECL biosensor system was successfully constructed. This method has many advantages: first, Au closed BPE provided the excellent conductivity than the graphite electrode. Second, the closed bipolar electrode system is a good choice compared with the three-electrode system, because it can distinguish the anode and cathode into two separate compartments, avoid any inter ference and have higher current efficiency. Third, AuPd NPs as a catalyst
Fig. 4 is a schematic diagram of the detection of miRNA-155 by the ECL method. Under the optimum conditions (see Fig. 3 and Supple mentary Material Fig. S3), the concentration of miRNA-155 could be reflected by ECL intensity. As shown in Fig. 4B, the logarithm of the concentration of miRNA-155 was proportional to the ECL intensity. Here different concentrations of miRNA-155 were selected to make standard
Fig. 3. (A) Optimization of electrical conductivity of BPE (a) Au NPs were all grown in the BPE region, (b) the BPE area is screen-printed with graphite electrodes loaded with gold at both ends, (c) screen printed graphite electrode in BPE area. (B) influence of the concentration of Ru(bpy)2þ 3 . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 5
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Fig. 4. (A) ECL responses of the biosensor with different concentrations of miRNA-155: 1 mM, 0.1 mM, 0.01 mM, 1 μM, 0.1 μM, 0.01 μM, 1 nM, 0.1 nM, 0.01 nM, 1 pM; under the condition of PBS (0.1 M, pH 7.0) including 0.5 nM H2O2. (B) The curve of ECL intensity vs the logarithm of miRNA-155 concentration.
Fig. 5. (A) The specificity of this proposed BPE-ECL biosensor for different interfering substance: (a) blank solution (0 fM miRNA-155), (b) let-7a (2 μM), (c) miRNA210 (2 μM), (d) sRNA (2 μM), (e) nRNA (2 μM), (f) miRNA-155 (2 μM). (B) The stability for different storage conditions of the proposed BPE-ECL biosensor (a) in a freezer, (b) in a refrigerator and (c) at room temperature.
could catalyze the reduction of H2O2 for signal amplification. Moreover, compared with other methods, the Au closed bipolar electrode electro chemiluminescence biosensor has good sensitivity and selectivity to miRNA-155. Therefore, this principle not only greatly expands the application of high performance BPE, but also provides a novel avenue for the detection of miRNA in clinical application, which can be applied other analytes.
items of University” of Jinan (2018GXRC001); the Taishan Scholars program and Case-by-Case Project for Top Outstanding Talents of Jinan. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.bios.2019.111917.
Declaration of competing interest
References
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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CRediT authorship contribution statement Fangfang Wang: Conceptualization, Methodology, Writing - orig inal draft. Cuiping Fu: Investigation, Formal analysis. Chuan Huang: Visualization, Data curation. Na Li: Resources, Visualization. Yanhu Wang: Writing - review & editing, Resources. Shenguang Ge: Conceptualization, Supervision, Funding acquisition. Jinghua Yu: Funding acquisition, Project administration. Acknowledgements This work was supported by the National Natural Science Foundation of China (21775055, 21575051, 21874055); Excellent Youth Innovation Team in Universities of Shandong (2019KJC016); The Project of “20 6
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