An electrochemical microRNA biosensor based on protein p19 combining an acridone derivate as indicator and DNA concatamers for signal amplification

Electrochemistry Communications 60 (2015) 185–189

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Short Communication

An electrochemical microRNA biosensor based on protein p19 combining an acridone derivate as indicator and DNA concatamers for signal amplification Chunyan Li a,1, Zhijing Liu b,1, Shuxian Cai b, Fadi Wen b, Dongzhi Wu b, Yingxin Liu b, Fang Wu a, Jianming Lan a, Zhizhong Han a, Jinghua Chen b,⁎ a

Department of Basic Chemistry, The School of Pharmacy, Fujian Medical University, Fuzhou, Fujian Province 350108, PR China Department of Pharmaceutical Analysis, The higher educational key laboratory for Nano Biomedical Technology of Fujian Province, The School of Pharmacy, Fujian Medical University, Fuzhou, Fujian Province 350108, PR China

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Article history: Received 17 August 2015 Received in revised form 7 September 2015 Accepted 9 September 2015 Available online 25 September 2015 Keywords: Electrochemical microRNA biosensor 5 7-dinitro-2-sulfo-acridone p19 protein-functionalized magnetic beads DNA concatamers

a b s t r a c t The new acridone derivative 5, 7-dinitro-2-sulfo-acridone (DSA) with excellent electrochemical activity was synthesized and reported for the first time in this paper. Then an electrochemical biosensor was fabricated for the signal amplified detection of microRNA (miRNA) via applying home-made DSA as signal unit. The p19 proteinfunctionalized magnetic beads (PFMBs) for specific recognition and enrichment of miRNA. Then DSA is combined with the long DNA concatamers, which functions as a signal enhancement platform to facilitate the high selectivity and sensitivity determination of miRNA. The usage of this novel electrochemical activity made a contribution to the performance of the approach, such as achieving a detection limit of 6 aM. To the best of our knowledge, this is the first attempt to apply DSA, PFMBs and long DNA concatamers for the fabrication of the electrochemical biosensors, which may represent a promising path toward early diagnosis of cancer at the point of care. © 2015 Elsevier B.V. All rights reserved.

1. Introduction MiRNAs are a group of noncoding RNAs (19–24 bases), which play important roles in a number of biological processes [1–4]. Numerous studies indicated it could be used to diagnose the diseases more effectively and utilized as minimally invasive biomarkers [5–7]. Therefore, it is urgently needed to develop methods for the miRNA analysis with high specificity and sensitivity. To date, many methods for miRNA detection have been proposed including microarray-based detection [8], capillary electrophoresis [9], and northern blot analysis [10]. Although a high sensitivity can be achieved, these methods still have some disadvantages such as high assay costs, time consuming step, and low detection accuracy. In an attempt to solve the above limitations, many electrochemical biosensors on the basis of signal amplification strategies were proposed. For example, Gao et al. presented an electrochemical biosensor for amplified miRNA detection based on hybridized miRNA-templated deposition of an insulating polymer film [11]. And other groups reported amplification methods based on endonuclease cleavage cycles [12], rolling circle

⁎ Corresponding author. Tel.: +86 591 22862016. E-mail address: [email protected] (J. Chen). 1 Zhijing Liu and Chunyan Li make equal contribution to the paper.

http://dx.doi.org/10.1016/j.elecom.2015.09.012 1388-2481/© 2015 Elsevier B.V. All rights reserved.

amplification (RCA) [13], and hybridization chain reaction [14]. However, most of these methods still require extraction of total RNA from real samples, which make the methods complicated, labor-intensive, and false positive results. Surprisingly, the Carnation Italian Ringspot Virus (CIRV) p19 protein (a tombusvirus protein of 19 kDa can interact with double-stranded RNA (dsRNA) structure of 21–23 nucleotides in length instead of binding to ssRNA, rRNA, ssDNA, dsDNA or mRNA [15].) was introduced into as a biorecognition element [4,5,15–21], which exhibits unique binding properties for specific enrichment and can decrease complexity, allowing to detect miRNAs with high selectively [19–21]. For example, Kilic et al. designed an electrochemical biosensor using the p19 as a molecular caliper for the first time for detection of miR-21 as low as in picomole sensitivity [4]. Campuzano et al. and Torrente-Rodríguez et al. demonstrated amperometric magnetobiosensors involving RNA-binding viral protein p19, achieving detection limit down to 40 pM and 0.61 nM, respectively [20,21]. Nevertheless, these methods have several deficiencies, such as laborious labeling techniques or environmentally sensitive, which hinder them from more extensive applications. Based on these facts, our group synthesized a novel acridone derivative DSA (see Fig. 1A) as a electrochemical indicator. Combination with the advantages of long DNA concatamers for signal amplification and PFMBs specific enrichment strategy, our proposed sensor may represent a promising path toward miRNA detection.

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Fig. 1. (A) Synthesis route of DSA. (B) Schematic representation of the electrochemical biosensor.

2. Experimental section 2.1. Reagents Acridone (A.R.) was purchased from Sigma. Salmon sperm DNA was purchased from Aldrich. The miRNA and DNA sequences were synthesized by TaKaRa Biotechnology Co. (Dalian, China). Their base sequences were as follows: Janus probe: 5′-UCA ACA UCA GUC UGA UAA GCU AAA CAT GAT GAC GGC C-3′; miR-21: 5′-UAG CUU AUC AGA CUG AUG UUG A-3′; auxiliary probe (AP1): 5′-GCA CCT GGG GGA GTA AGT GGC CGT CAT CAT-3′; auxiliary probe (AP2): 5′-ACT CCC CCA GGT GCA TGA TGA CGG CCA CT-3′; single-base mismatched miRNA (MT1): 5′-UAG CUU AUC ACA CUG AUG UUG A-3′; single-base mismatched miRNA (MT2): 5′-UAG CUU AUC AGA CUG AUG UUC A-3′; single-base mismatched miRNA (MT3): 5′-UAC CUU AUC AGA CUG AUG UUG A-3′; non-complementary sequence (NC): 5′-AGA GGT UGU

TUG UCC CAU AGU C-3′. P19 protein, chitin magnetic beads and magnetic separation rack were purchased from New England BioLabs. All other reagents were of analytical reagent grade. All solutions were prepared with MilliQ water (18.2 MΩ). 2.2. Apparatus CHI 660E Electrochemical Workstation (Shanghai CH Instruments, China) was used for electrochemical measurements with the magnetic glassy carbon electrode (MGCE) (Tianjin Gaoss Union, China) as the working electrode, a Ag/AgCl as reference electrode and a platinum wire as counter electrode. Perkin-Elmer Spectrum 2000 FT-IR spectrometer (USA). Elementarvario EL III elemental analyzer. Thermo Finnigan DECAX-30000 LCQ Deca XP ion trap mass spectrometry. Bruker Avance III HD 400 M nuclear magnetic resonance analyzer. For square wave voltammetrys (SWVs) scanning, the potential scanning range was

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from −0.20 V–+0.15 V; square wave frequency was 10 Hz; the square wave amplitude was 50 mV and scan potential increment was 2 s. 2.3. The preparation of DSA As shown in Fig. 1A, 2-sulfo-acridone (Fig. 1A-1) was synthesized starting with 9(10H)-acridone. An amount of 0.6 g acridone was put into a beaker, 5 mL H2SO4 was added then, and the temperature was raised to 100 °C. Then the reaction was performed for 30 min under stirring. The solution was poured into ice water and solid precipitate appeared. The resulting solid was filtered off, washed with a few water and further purified by recrystallization in absolute alcohol. The solid was then oven-dried at 75 °C and yellow crystal of 1 (Fig. 1A-1) could be obtained. DSA (Fig. 1A-2) was synthesized starting with 1. An amount of 1 of 0.6 g was put into a beaker and 3 mL of 36% acetic acid, 0.36 mL nitric acid and 0.8 mL glacial acetic acid were added, the temperature was raised to 58 °C and the reaction was performed for 2 h under stirring. The solution was poured into ice water and solid precipitate appeared. The resulting solid was filtered off, washed with a few water and further purified by recrystallization in absolute alcohol. The solid was then oven-dried at 80 °C and yellow crystal of 2 (Fig. 1A-2) could be obtained, the yield was 75%. Analytical for C13H7N3O8S: C, 42.83%; H, 1.99%; N, 11.52% (calculated: C, 42.75%; H, 1.93%; N, 11.50%). The infrared spectrum of DSA was examined, and IR (KBr) υ: 3410 (υNH), 1592 (υC-C), 1642 (υC = C), 1389 (υC-NO2), 1190 (υSO2OH). FAB-MS: m/z 366 ([M + 1]+). 1H NMR (CDCl3, δ): 9.04 (s, ArH), 8.76 (s, ArH), 8.13 (s, ArH), 7.85 (d, ArH), 6.87 (d, ArH), 4.21 (s, NH). 13C-NMR (100 MHz, DMSO) δ: 124.21 (_CH −), 128.98 (_CH −), 124.88 (_CH −), 132.21 (_CH −), 122.11 (_CH −), 123.96 (_C–C_O), 123.87 (_C–C_O), 142.70 (_C–N), 142.78 (_CN), 176.70 (–C_O), 131.57 (_C–SO3H), 137.92 (_C–NO2), 141.21 (_C–NO2). 2.4. The preparation of electrochemical biosensor First of all, hybridization of the Janus probe and target miR-21 to form dsRNA was performed in incubator with constant temperature and humidity for 2 h at 30 °C. At the same time, p19 protein was immobilized on the surface of magnetic beads to form a stable complex, PFMBs. Next, 5 μL of PFMBs was dropped on the surface of MGCEs to form PFMBs/MGCEs. Then, the modified PFMBs electrode was immersed in the above 0.5 mL of Janus probe-miRNA complex. The binding reaction was performed by shaking for 2 h at room temperature. Thus with the purpose of removing the non-specific absorption, they were washed three times with 500 μL of washing buffer (20 mM Tris–HCl, 100 mM NaCl, 1 mM EDTA, 100 μg/mL BSA) and one time in 500 μL MilliQ water. Subsequently, 5 μL of freshly prepared DNA hybridization buffer (10 mM Tris–HCl containing 1 mM EDTA, 500 mM NaCl and 10 mM MgCl2, pH 7.4) containing 1.0 μM AP1 and 1.0 μM AP2 was dropped on the surface of the electrodes, incubated for 2 h and hybridization with Janus probe. Afterwards, the modified electrode was immersed into 1.0 mM DSA solution dissolving in pH 6.0 PB buffer for 5 min at room temperature. Then, the electrodes were rinsed with the same PB buffer and water successively. Finally, the electrochemical investigation was carried out in a 10 mL electrochemical cell. 3. Results and discussion 3.1. Experimental principle of the electrochemical biosensor The detailed conceptually fundamentals of the sensor was illustrated in Fig. 1B. We designed a bifunctional Janus probe whose sequence contains two regions, including the complementary RNA oligonucleotide sequence of miR-21 (marked in green in Fig. 1B) and DNA oligonucleotide sequence used as primer for long-range self-assembled DNA concatamers (marked in yellow in Fig. 1B). In the presence of miR-21,

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the Janus probe preferred to form dsRNA which was accompanied by specific binding between the PFMBs and dsRNA. Thus, the separation and enrichment of miR-21 anchored on PFMBs was achieved on the surface of MGCE. Subsequently, two auxiliary probes (AP1 and AP2) were added to the solution, and thus formed long DNA concatamers [22,23]. In this case, myriad DSAs could bind to the dsDNA structures through intercalation, resulting in an intense electrochemical response. Nevertheless, in the absence of miR-21, it could not form dsRNA. In this state, the DNA concatamers could not be anchored to MGCE. Therefore, there would have no the intercalative binding effect of DSA to dsDNA, resulting in a poor electrochemical signal. Electrochemical impedance spectrum (EIS) was employed to characterize the fabrication in whole process. The results indicated that our biosensor indeed worked as expected (data were not shown in).

3.2. The sensitivity of the biosensor To investigate the sensitivity of the biosensor, the current signals were recorded upon addition of the miR-21 at varying concentrations by SWV techniques. As shown in Fig. 2A, the current signals increased with the increasing concentrations of miR-21 from 20 aM–100 aM (as shown in the inset). The correlation equation is: I (μA) = (0.0287 ± 0.003)C(aM) − (0.0331 ± 0.1955), where the errors denote 95% confidence intervals and a correlation coefficient of 0.9933 could be obtained. The limit of detection (LOD) was calculated to be 6 aM based on 3σ method. The relative standard deviation (RSD) for 3 repetitive measurements in the same electrode with the same set of reagents of 100 aM miR-21 was 6.20%. Also, the current signals of different electrodes were tested, and the RSD was 9.48%. Compared with several previous electrochemical sensors methods including using protein p19 [4,20, 21] (the LODs of ref. [4,20,21] were 0.16 μM, 40 pM and 0.61 nM, respectively) used for miRNA determination, the achieved LOD of our method was lower. Our proposed method possesses better sensitivity because it using DSA, the biosensor required no the redox-labeled DNA as a signal transducer, which not only decreases the background signal by preventing the labeling moieties from interfering with DNA binding but also simplifies the fabrication of the sensors. Specifically, the specific recognition enrichment of PFMBs and signal amplification from long DNA concatamers made a contribution to the excellent sensitivity.

3.3. The selectivity of the sensor The specificity of the proposed biosensor was investigated through a comparison assay on mismatch targets and perfect complementary target, including complementary target, different single-base mismatched strands, and non-complementary strand at a same concentration. As Fig. 2B illustrated, the current of a perfectly complementary target was higher than that of MT1–MT3 (approximately 9.0, 7.5, 6.7 times, respectively), and the response of the non-complementary strand was almost ignored. These results suggested that the sensor owned high sequence specificity and excellent discrimination for similar miRNAs. The direct detection of the expression level of miR-21 in the human blood serum extracted from patients was explored to evaluate the applicability and explore the feasibility of our biosensor. MiR-21 in the human blood serum from three types of cancer patients, including breast cancer, lung cancer, and gastric cancer, was measured. The concentration of miR-21 in cancer patients is higher than that in the healthy group as shown in Fig. 2C, suggesting an upregulation miR-21 in the serum of cancer patient. Interestingly, the miR-21 concentration of sample 4 (breast cancer patient, about 1.2 fM, which is consistent with the previous report [24]) is even 37.5-fold compared with sample 1 (healthy person, about 32 aM). These results demonstrated that our biosensor could be applied to the direct determination of miR-21 in serum samples without separation.

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DSA can be easily prepared with high yield using inexpensive materials, which make our method a favorable universality. (2) The specific enrichment and the efficient magnetic separation of PFMBs have been extremely useful for discriminating against unwanted constituents, including a large excess of co-existing non-complementary and mismatched RNA, chromosomal DNA, and proteins. (3) The long DNA concatamers can carry enormous DSA, which results in the significantly enhanced electrochemical signal. (4) The “signals on” strategy can decrease the rate of the false-negative and the false-positive. In summary, due to its high sensitivity and selectivity, easy-to-use, fast and low cost, the proposed biosensor allows for the integration into portable systems and on-site cancer screening applications. Conflict of interest

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The authors declare no competing financial interest.

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The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (21375017, 21105012), the National Science Foundation for Distinguished Young Scholars of Fujian Province (2013J06003), the National Science Foundation of Fujian Province (2015J01596, 2015J05020), the Key Project of Fujian Science and Technology (2013Y0045), Program for Fujian University Outstanding Youth Scientific Research (JA11105, JA10295), Program for New Century Excellent Talents of Colleges and Universities in Fujian Province (JA13130), the Foundation of Fuzhou Science and Technology Bureau (2013-S-122-4), the Medical Elite Cultivation Program of Fujian Provincial Health and Family Planning Commission (2014-ZQN-ZD-26), the Foundation of Fujian Provincial Department of Education (JA13149), Youth Scientific Research Program of Fujian Provincial Health and Family Planning Commission (2014-1-39) and Nursery Scientific Research Foundation of Fujian Medical University (2014MP008).

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Fig. 2. (A) Typical SWV curves on the bare electrode (curve a) and upon addition of different concentrations of miR-21 (0, 20 aM, 30 aM, 40 aM, 50 aM, 60 aM, 70 aM, 80 aM, 90 aM, 100 aM.) (curves b–k, respectively). Inset: linear relationship between the current intensity and the concentration of miRNA. The concentration of Janus probe, MBs, AP1 and AP2 were 20 nM, 0.2 mg/mL, 1.0 μM and 1.0 μM, respectively. Each data point represents the average value of three independent experiments with error bars indicated. (B) SWV signals a) of the blank and in the presence of b) NC, c) MT1, d) MT2, e) MT3, and f) miR21. All miRNAs were applied at a 100 aM concentration for the assay. The concentration of Janus probe, MBs, AP1 and AP2 were 20 nM, 0.2 mg/mL, 1.0 μM and 1.0 μM, respectively. (C) SWV signals of miR-21 in human blood serum obtained by electrochemical biosensor. Samples 1–3 were from three healthy persons. Samples 4 and 5 were from two breast cancers patients. Samples 6 and 7 were from two lung cancer patients. Samples 8 and 9 were from two gastric cancer patients. The concentration of Janus probe, MBs, AP1 and AP2 were 20 nM, 0.2 mg/mL, 1.0 μM and 1.0 μM, respectively. Each value is the average of three measurements.

4. Conclusions In this work, a sensitive and specific electrochemical biosensor for miR-21 assay was developed based on protein-facilitated specific enrichment strategy and home-made DSA as electrochemical hybridization indicator. The incorporation of DSA, PFMBs and long DNA concatamers in the biosensor has resulted in many advantages. (1) The excellent identification capability of DSA between dsDNA and ssDNA, which not only reduce the background signal but also simplify the fabrication of the biosensor. Meanwhile, the small molecule of

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