Label-free fluorescence strategy for sensitive microRNA detection based on isothermal exponential amplification and graphene oxide

Author’s Accepted Manuscript Label-free fluorescence strategy for sensitive microRNA detection based on isothermal exponential amplification and graphene oxide Wei Li, Ting Hou, Min Wu, Feng Li www.elsevier.com/locate/talanta

PII: DOI: Reference:

S0039-9140(15)30444-6 http://dx.doi.org/10.1016/j.talanta.2015.10.078 TAL16086

To appear in: Talanta Received date: 21 September 2015 Accepted date: 25 October 2015 Cite this article as: Wei Li, Ting Hou, Min Wu and Feng Li, Label-free fluorescence strategy for sensitive microRNA detection based on isothermal exponential amplification and graphene oxide, Talanta, http://dx.doi.org/10.1016/j.talanta.2015.10.078 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Label-free fluorescence strategy for sensitive microRNA detection based on isothermal exponential amplification and graphene oxide Wei Li, Ting Hou, Min Wu, Feng Li* College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University Qingdao 266109, China

Submitted to Talanta, September 21, 2015 *Corresponding author. E-mail: [email protected]; Tel/Fax: +86-532-86080855 Abstract MicroRNAs (miRNAs) play an important role in many biological processes, and have been regarded as potential targets and biomarkers in cancer diagnosis and therapy. Also, to meet the big challenge imposed by the characteristics of miRNAs, such as small size and vulnerability to enzymatic digestion, it is of great importance to develop accurate, sensitive and simple miRNA assays. Herein, we developed a label-free fluorescence strategy for sensitive miRNA detection by combining isothermal exponential amplification and the unique features of SYBR Green I (SG) and graphene oxide (GO), in which SG gives significantly enhanced fluorescence upon intercalation into double-stranded DNAs (dsDNAs), and GO selectively adsorbs miRNA, single-stranded DNA and SG, to protect miRNA from enzymatic digestion, and to quench the fluorescence of the adsorbed SG. In the presence of the target miRNA, the ingeniously designed hairpin probe (HP) is unfolded and the subsequent polymerization and strand displacement reaction takes place to initiate the target recycling process. The newly formed dsDNAs are then recognized and cleaved by the nicking enzyme, generating new DNA triggers with the same sequence as the target miRNA, which hybridize with intact HPs to initiate new extension reactions. As a result, the circular exponential amplification for target miRNA is achieved and large amount of dsDNAs are formed to generate significantly enhanced fluorescence upon the intercalation of SG. Thus sensitive and selective fluorescence miRNA detection is realized, and the detection

1

limit of 3 fM is obtained. Besides, this method exhibits additional advantages of simplicity and low cost, since expensive and tedious labelling process is avoided. Therefore, the as-proposed label-free fluorescence strategy has great potential in the applications in miRNA-related clinical practices and biochemical researches. Keywords: Label free; Fluorescence; MicroRNA; Isothermal exponential amplification; Graphene oxide

1. Introduction MicroRNAs (miRNAs), a class of single-stranded, small (containing approximately 19 to 23 nucleotides) and non-coding RNAs found in eukaryotic cells [1], can regulate gene expression by binding to messenger RNAs (mRNAs), which further influence the biological processes [2]. Up till now, over 1000 human miRNAs have been identified, which can target more than 30% of the human genome [3]. Apart from acting as regulators in gene expression, abnormal expressions of certain miRNAs are closely related to a variety of diseases and disorders, such as cancers, cardiovascular and autoimmune diseases [4], so miRNAs have been regarded as potential targets in disease diagnosis and therapy, as well as new biomarkers for many diseases, especially cancers [5,6]. Thus, sensitive and selective miRNA detection is highly desirable in the fields of biomedical research and early clinical diagnosis. MiRNA detection is facing challenge due to the unique characteristics of miRNA: (1) the low cellular abundance of miRNA that requires the method to be highly sensitive, (2) the highly homogenous sequences with as few as one-base difference in miRNA family that requires high specificity of the method, (3) the intrinsic vulnerability to enzymatic digestion that requires strict environment for miRNA analysis, (4) other characteristics of miRNA such as the short length also aggravate the difficulty in miRNA detection [7,8]. Conventional methods, including Northern blotting [9], real-time PCR [10], and microarrays [11] have been widely used in miRNA analysis. However, these methods have the limitations of low sensitivity, poor specificity and long assay time. To overcome these shortcomings, several signal amplification strategies have been adopted for miRNA detection [12–27], including hybridization chain reaction [15,20], rolling circle amplification [12,22], and strand displacement assay [16]. Among them, isothermal exponential amplification has attracted much attention in miRNA detection due to its high sensitivity, good specificity and simplicity [23–27]. For instance, Shi’s group reported a hairpin fluorescence probe-assisted isothermal exponential

2

amplification strategy [24]. Zhang’s group reported a hairpin probe-based circular exponential amplification assay for sensitive detection of miRNAs [25]. However, these assays need the fluorophore and quencherlabelled molecular beacons, inevitably causing expensive and tedious operations. Thus it is of great significance to realize label-free isothermal exponential amplification for miRNA analysis. Graphene oxide (GO) has emerged as an excellent platform for biological applications due to its unique characteristics, such as water-solubility and versatile surface modification [22,28–31]. Among the GO-based sensing strategies, DNA optical sensors have attracted much attention owing to GO’s selective adsorption of single-stranded DNAs (ssDNAs) via the non-covalent π−π stacking interactions and its superior fluorescence quenching ability [20,32–40]. For example, Li’s group reported GO surface-anchored fluorescence sensor for sensitive detection of miRNA coupled with enzyme-free signal amplification of hybridization chain reaction [20]. Our group reported an enzyme-free and label-free fluorescence aptasensing strategy for highly sensitive detection of protein based on target-triggered hybridization chain reaction amplification [35]. Very recently, Tang’s group reported that GO can efficiently protect miRNA from being digested by RNase [33]. In consideration of its functions of protecting miRNA from enzymatic digestion and selective adsorption of ssDNA, we believe that GO is a good candidate to enhance the sensitivity of fluorescence miRNA detection. Herein, we present a novel label-free fluorescence strategy based on isothermal exponential amplification for miRNA detection by using SYBR Green I (SG) as the signal readout [20,35] and GO as the protector, adsorber and quencher. In the absence of the target miRNA, the hairpin probe (HP), the primer and SG are adsorbed on GO and the fluorescence is quenched to result in a very low background signal. However, in the presence of the target miRNA, HP is unfolded and the subsequent polymerization and strand displacement reaction take place to initiate the target recycling process. The newly formed double-stranded DNAs (dsDNAs) are then recognized and cleaved by the nicking enzyme, generating new DNA triggers with the same sequence as the target miRNA, which hybridize with intact HPs to initiate new extension reactions. As a result, the circular exponential amplification for target miRNA is achieved and large amount of dsDNA are formed. Upon the intercalation of SG into dsDNAs, a significantly enhanced fluorescence signal is obtained, endowing this GO-assisted label-free fluorescence miRNA assay with high sensitivity and good selectivity.

3

2. Experimental 2.1. Reagents and materials The HPLC-purified miRNA and diethylprocarbonate (DEPC)-treated deionized water were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). Nb.BtsI and RNase were purchased from New England Biolabs, Ltd. (Beijing, China). HPLC-purified oligonucleotides, Klenow fragment (KF) polymerase (without 3' to 5' exonuclease activity), ribonuclease inhibitor, and deoxyribonucleoside triphosphates (dNTPs) were purchased

from

Shanghai

Sangon

Biotechnology

Co.,

Ltd.

(Shanghai,

China).

Tris(hydroxymethyl)aminomethane (Tris), hydrochloric acid (HCl), dithiothreitol (DTT), MgCl2, NaCl, KAc and Mg(Ac)2 were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), which were of analytical grade and used without further treatment. SYBR Green I (SG, 10000 ×) was purchased from SigmaAldrich Co. LLC. (St. Louis, MO, USA). Graphene oxide was purchased from Nanjing XFNANO Materials Co. Ltd. (Nanjing, China). The sequences of the oligonucleotides were listed in Table 1. All oligonucleotides were used as provided, and miRNAs were diluted with DEPC-treated water and DNA oligonucleotides were diluted with 10 mM Tris-HCl buffer (pH 7.9, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT) to give the stock solutions. DEPC-treated deionized water was used throughout the experiments. <>

2.2. Instrument All fluorescence measurements were performed a Hitachi F-4600 fluorescence spectrophotometer (Japan) under room temperature. The excitation wavelength was set to 493 nm, and the 24 photomultiplier tube voltage was set to 700 V. The emission spectra from 510 to 580 nm were collected, with the maximum emission observed at 520 nm. The slits for excitation and emission were both set to 5 nm. To minimize the effect of RNase on the stability of miRNAs, all glassware, pipette tips and centrifuge tubes were autoclaved using Tomy SX-500 Autoclave (Tokyo, Japan).

2.3. Isothermal exponential amplification reaction Before the reaction, HP was incubated at 95 °C for 5 min and then cooled to room temperature over 30 min

4

to make the probe perfectly fold into a hairpin structure. The amplification reaction mixture with a total volume of 90 μL contained 1.8 μg of GO, 25 nM HP, 25 nM primer, 0.5 mM dNTPs, 12 U of KF polymerase, 10 U of Nb.BtsI, 18 U of RNase inhibitor, 1 × KF polymerase buffer (pH 7.9, 50 mM Tris−HCl, 5 mM MgCl2, 1 mM DTT), 1 × Nb.BtsI buffer (pH 7.9, 50 mM Tris−HAc, 50 mM KAc, 10 mM Mg(Ac)2, 100 µg/mL BSA) and the target miRNA (miRNA21) with different concentrations, and this reaction mixture was incubated at 37 °C for 2 h. The amplification product was then incubated with 10 μL of 20 × SG at 37 °C for 10 min before fluorescence measurements. All experiments were repeated three times.

3. Results and discussion 3.1. Principle of label-free fluorescence miRNA assay The principle of the label-free fluorescence strategy for miRNA detection is illustrated in Scheme 1. A hairpin probe (HP) is ingeniously designed, which composed of four DNA domains: (1) a domain complementary to the target miRNA, (2) a domain complementary to the primer, (3) a domain complementary to the recognition sequences of the nicking enzyme, and (4) a dangling single-stranded domain helping HP to be fully adsorbed onto GO to further decrease the background signal. In this experiment, firstly, GO is added to the target miRNA solution and miRNA is adsorbed on the surface of GO via π-π stacking and effectively protected from enzymatic digestion. Then, with HP and the primer being added to the reaction solution, miRNA unfolds HP to expose the domain complementary to the primer, which subsequently hybridizes with the primer to initiate the polymerization process under the assistance of KF polymerase and dNTPs. The elongated DNA strand thus displaces the target miRNA, forming a long dsDNA structure. The released target miRNA then hybridizes with an intact HP to initiate the target recycling amplification process. Moreover, the nicking endonuclease (Nb.BtsI) recognizes the duplex site and cleaves the elongated DNA strand, to initiate the subsequent polymerization and strand displacement cycling process, generating a large number of DNA triggers (tDNA) with the same sequence as the target miRNA, and tDNA is more stable than miRNA and can initiate a new extension reaction. By following this mechanism, a new circular exponential amplification for the target miRNA is achieved. Upon the addition of SG to the reaction solution, a highly enhanced fluorescence signal is obtained due to the selective intercalation of SG into dsDNA detached from the GO surface [41]. However, in the absence of the target miRNA, HP maintains the hairpin structure, with the

5

primer-complementary sequences embedded in its stem, so the 3¢-phosphorylated end of HP prevents the KF polymerase-catalyzed polymerization. Thus the two aforementioned cycling amplification processes cannot occur, and no dsDNA is formed. In addition, since HP is adsorbed on the surface of GO via its long singlestranded segment, the fluorescence derived from the SG inserted into the stem of HP is efficiently quenched by GO, resulting in a small fluorescence background signal. Thus sensitive label-free fluorescence miRNA assay based on isothermal exponential amplification is readily realized. <>

3.2. Feasibility study of miRNA assay The proof-of-concept experiments were carried out to investigate the feasibility of the proposed strategy for miRNA assay. First, the adsorption ability of GO toward DNA and SG was tested. It has been reported that ssDNA can be strongly adsorbed on the surface of GO via π-π stacking and the fluorescence of the fluorophore labeled on ssDNA is effectively quenched, whereas dsDNA shows less affinity toward GO [20,35]. Moreover, SG shows high fluorescence enhancement when it is intercalated into dsDNA, but little fluorescence in solution or in the presence of ssDNA [41]. As shown in Fig. 1A, SG in solution showed negligible fluorescence (curve a), and similar results were also observed in the presence of GO (curve b), GO and the target miRNA (miRNA21) (curve c), GO and an ssDNA (denoted as ssDNA1) with the same sequence as miRNA21 (curve d). A slightly enhanced fluorescence intensity was detected when SG was added in the solution containing both GO and HP (curve e), which may be due to the fact that the long single-stranded domain of HP facilitated the adsorption of HP on the surface of GO to quench the fluorescence of SG intercalated into the duplex stem of HP. However, a high fluorescence signal was observed when SG, GO and dsDNA (i.e. the hybridization product of ssDNA1 and its complementary DNA sequences, denoted as ssDNA2) were present (curve f), which clearly demonstrated that GO showed little affinity to dsDNA, thus when SG was intercalated into dsDNA, its fluorescence could not be quenched by GO. One of the big challenges for the detection of miRNAs is their vulnerability to enzymatic digestion. Recently, Tang’s group reported that GO can effectively protect miRNA from enzymatic digestion [33]. So in this experiment, we also investigated the protection ability of GO for miRNA. As shown in Fig. 1B, when

6

miRNA21 was treated with RNase for 1 h, after the inactivation treatment and the addition of its complementary DNA sequences (ssDNA2) and SG, a small fluorescence signal was observed (column a). However, if GO was present in the above reaction system, a high fluorescence signal was observed (column b). Fluorescence with similar intensity was also obtained in the presence of RNase inhibitor no matter GO was added (column d) or not (column c). These results suggested that miRNA was protected from RNase digestion after being adsorbed on the surface of GO. So, the introduction of GO into the miRNA detection system will enhance the stability of miRNA and further improve the accuracy and sensitivity of the miRNA assay. Finally, the feasibly of the as-proposed miRNA assay was investigated. As shown in Fig. 1C, in the presence of GO and HP, a small fluorescence signal was observed (curve a), suggesting that HP and SG were effectively adsorbed on the surface of GO to quench the fluorescence. With the primer (Primer2), KF polymerase and dNTP added into the reaction system, only a slight fluorescence enhancement was observed (curve b), and with Nb.BtsI further added into the system, the fluorescence intensity barely changed (curve c), demonstrating that in the absence of the target miRNA, no dsDNA was formed and the exponential amplification process did not occur. However, once the target miRNA (miRNA21) was added to the reaction system, a significant fluorescence enhancement was observed (curve d), indicating that the circular signal amplification for target miRNA detection indeed took place. Therefore, the as-proposed strategy is feasible for sensitive detection of miRNA. <>

3.3. Optimization of experimental conditions To get the best performance of the proposed assay for miRNA, the primer length, HP concentration and KF polymerase amount were optimized. The length of the primer is a crucial parameter. If the primer is too short, it is incapable of hybridizing with HP, thus resulting in no fluorescence gain upon the addition of the target miRNA. However, if the primer is too long, it is possible for the primer to unfold HP and increase the background signal. Thus the fluorescence responses in the presence of 6, 7, 8 or 9-mer primers (denoted as Primer1, Primer2, Primer 3 and Primer 4, respectively) were investigated, respectively. As shown in Fig. 2A, the relative fluorescence intensity, i.e. (F–F0)/F0, in which F0 and F were the fluorescence intensity in the absence and presence of miRNA21, respectively, increased as the length of the primer increased from 6-mer to

7

7-mer, then decreased as the primer length further increased to 8-mer and 9-mer. Therefore, 7-mer was chosen as the optimal length for the primer and thus Primer2 was used in the subsequent experiments. In addition, HP concentration and KF polymerase amount were optimized. As shown in Fig. 2B, the fluorescence signal increased gradually with the increase of HP concentration and then reached a stabilized platform at the HP concentration of 25 nM. As illustrated in Fig. 2C, as the KF amount increased from 4 to 20 U, the fluorescence intensity initially increased and leveled off after KF polymerase reached 12 U. Therefore, 25 nM HP and 12 U of KF polymerase were chosen as the optimal experimental conditions and used in the subsequent experiments. <>

3.4. Analytical performance of miRNA assay Under the optimal experimental conditions, miRNA21 with different concentrations was added into the reaction system to evaluate the analytical performance of this sensing platform. As illustrated in Fig. 3, the fluorescence intensity gradually increased with the miRNA21 concentration increased from 0 to 10 pM, which was in accordance with the fact that miRNA with higher concentration would induce more dsDNA formation with the assistance of the primer, KF polymerase and nicking enzyme Nb.BtsI. The inset of Fig. 3 showed a good linear correlation between F-F0 (in which F and F0 are the fluorescence intensity in the presence and absence of miRNA21, respectively) and the logarithm of miRNA21 concentration ranging from 10 fM to 10 pM. The correlation equation was determined to be (F-F0) = 387.92 lgC - 184.92 with a correlation coefficient of R2 = 0.9915, and the detection limit was estimated to be 3 fM (based on 3s). The results demonstrated that sensitive detection of miRNA was indeed realized by the as-proposed label-free fluorescence exponential amplification strategy. In addition, the reproducibility of the miRNA biosensing platform was investigated through 5 successive assays in the presence of 500 fM miRNA21, and the relative standard deviation (RSD) was determined to be 3.58%, indicating an acceptable repeatability of the asproposed strategy. <>

3.5. Selectivity of miRNA assay The selectivity of this experiment was further investigated by adding miRNA21 and its analogs (miRNA210

8

and miRNA214) with the same concentration into the reaction system, respectively, in which HP containing a perfectly complementary segment to miRNA21 was adopted. As shown in Fig. 4, high fluorescence intensity enhancement was realized only when miRNA21 was present, whereas in the presence of miRNA210 or miRNA214, relatively small fluorescence signal was observed, which was comparable to that in the control experiment. Thus, the as-proposed miRNA detection approach exhibited high sequence specificity to discriminate target miRNA from its analogs in the same miRNA family. <>

4. Conclusions In summary, we have proposed a label-free fluorescence strategy for sensitive detection of miRNA by combining isothermal exponential amplification and the unique features of GO and SG, in which GO acts as the protector, adsorber and quencher, and SG as the signal readout. Through the cycling processes of hybridization, polymerization, enzymatic cleavage and strand displacement, small amount of target miRNA results in a large number of dsDNAs, generating significantly enhanced fluorescence upon the intercalation of SG. In addition, intact HPs can be adsorbed onto GO surface through its long single-stranded segment, thus the fluorescence of SG intercalated into the stem of HP is effectively quenched, resulting in a very low background signal. This biosensing strategy exhibits excellent selectivity and sensitivity for miRNA assay, as well as the additional advantages of simplicity and low cost, since expensive and tedious labeling process is avoided. Therefore, the as-proposed label-free fluorescence miRNA assay may become an alternative method for simple and sensitive miRNA detection in clinical diagnostics and biochemical researches.

Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 21175076, 21575074 and 21445002), Project of Shandong Province Higher Educational Science and Technology Program (J15LC08), and the Research Foundation for Distinguished Scholars of Qingdao Agricultural University (No. 663-1113311 and 663-1113320).

9

References [1] L. He, G.J. Hannon, MicroRNAs: small RNAs with a big role in gene regulation, Nat. Rev. Genet., 5 (2004) 522–531. [2] D.P. Bartel, MicroRNAs: target recognition and regulatory functions, Cell, 136 (2009) 215–233. [3] H.F. Dong, J.P. Lei, L. Ding, Y.Q. Wen, H.X. Ju, X.J. Zhang, MicroRNA: function, detection, and bioanalysis, Chem. Rev., 113 (2013) 6207–6233. [4] M.V. Iorio, R. Visone, G. Di Leva, V. Donati, F. Petrocca, P. Casalini, et al., MicroRNA signatures in human ovarian cancer, Cancer Res., 67 (2007) 8699–8707. [5] J.B. Li, S. Tan, R. Kooger, C.Y. Zhang, Y. Zhang, MicroRNAs as novel biological targets for detection and regulation, Chem. Soc. Rev., 43 (2014) 506–517. [6] J. Lu, G. Getz, E.A. Miska, E. Alvarez-Saavedra, J. Lamb, D. Peck, et al., MicroRNA expression profiles classify human cancers, Nature, 435 (2005) 834–838. [7] H.F. Dong, J.P. Lei, L. Ding, Y.Q. Wen, H.X. Ju, X.J. Zhang, MicroRNA: function, detection, and bioanalysis, Chem. Rev., 113 (2013) 6207–6233. [8] X.Y. Lin, C. Zhang, Y.S. Huang, Z. Zhu, X. Chen, C.Y.J. Yang, Backbone-modified molecular beacons for highly sensitive and selective detection of microRNAs based on duplex specific nuclease signal amplification, Chem. Commun., 49 (2013) 7243–7245. [9] A. Válóczi, C. Hornyik, N. Varga, J. Burgyán, S. Kauppinen, Z. Havelda, Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes, Nucleic Acids Res., 32 (2004) e175. [10] C. Chen, D.A. Ridzon, A.J. Broomer, Z.H. Zhou, D.H. Lee, J.T. Nguyen, et al., Real-time quantification of microRNAs by stem–loop RT–PCR, Nucleic Acids Res., 33 (2005) e179. [11] J.M. Thomson, J. Parker, C.M. Perou, S.M. Hammond, A custom microarray platform for analysis of microRNA gene expression, Nat Meth, 1 (2004) 47–53. [12] J.J. Yao, K. Flack, L.Z. Ding, W.W. Zhong, Tagging the rolling circle products with nanocrystal clusters for cascade signal increase in the detection of miRNA, Analyst, 138 (2013) 3121–3125. [13] H.V. Tran, B. Piro, S. Reisberg, H.T. Duc, M.C. Pham, Antibodies directed to RNA/DNA hybrids: an

10

electrochemical immunosensor for microRNAs detection using graphene-composite electrodes, Anal. Chem., 85 (2013) 8469–8474. [14] R.X. Duan, X.L. Zuo, S.T. Wang, X.Y. Quan, D.L. Chen, Z.F. Chen, et al., Lab in a tube: ultrasensitive detection of microRNAs at the single-cell level and in breast cancer patients using quadratic isothermal amplification, J. Am. Chem. Soc., 135 (2013) 4604–4607. [15] Z.L. Ge, M.H. Lin, P. Wang, H. Pei, J. Yan, J.Y. Shi, et al., Hybridization chain reaction amplification of microRNA detection with a tetrahedral DNA nanostructure-based electrochemical biosensor, Anal. Chem., 86 (2014) 2124–2130. [16] Y.H. Liao, R. Huang, Z.K. Ma, Y.X. Wu, X.M. Zhou, D. Xing, Target-triggered enzyme-free amplification strategy for sensitive detection of microRNA in tumor cells and tissues, Anal. Chem., 86 (2014) 4596–4604. [17] J.H. Liu, C.Y. Wang, Y. Jiang, Y.P. Hu, J.S. Li, S. Yang, et al., Graphene signal amplification for sensitive and real-time fluorescence anisotropy detection of small molecules, Anal. Chem., 85 (2013) 1424–1430. [18] Y.Q. Ren, H.M. Deng, W. Shen, Z.Q. Gao, A highly sensitive and selective electrochemical biosensor for direct detection of microRNAs in serum, Anal. Chem., 85 (2013) 4784–4789. [19] C. Shi, Q. Liu, C.P. Ma, W.W. Zhong, Exponential strand-displacement amplification for detection of microRNAs, Anal. Chem., 86 (2014) 336–339. [20] L. Yang, C.H. Liu, W. Ren, Z.P. Li, Graphene surface-anchored fluorescence sensor for sensitive detection of microRNA coupled with enzyme-free signal amplification of hybridization chain reaction, ACS Appl. Mater. Interfaces, 4 (2012) 6450–6453. [21] B.C. Yin, Y.Q. Liu, B.C. Ye, One-step, multiplexed fluorescence detection of microRNAs based on duplex-specific nuclease signal amplification, J. Am. Chem. Soc., 134 (2012) 5064–5067. [22] H.Y. Liu, L. Li, L.L. Duan, X. Wang, Y.X. Xie, L.L. Tong, et al., High specific and ultrasensitive isothermal detection of microRNA by padlock probe-based exponential rolling circle amplification, Anal. Chem., 85 (2013) 7941–7947. [23] Y.Q. Liu, M. Zhang, B.C. Yin, B.C. Ye, Attomolar ultrasensitive microRNA detection by DNA-scaffolded silver-nanocluster probe based on isothermal amplification, Anal. Chem., 84 (2012) 5165–5169. [24] C.P. Ma, S. Liu, C. Shi, Ultrasensitive detection of microRNAs based on hairpin fluorescence probe

11

assisted isothermal amplification, Biosens. Bioelectron., 58 (2014) 57–60. [25] G.L. Wang, C.Y. Zhang, Sensitive detection of microRNAs with hairpin probe-based circular exponential amplification assay, Anal. Chem., 84 (2012) 7037–7042. [26] D.B. Zhu, L. Zhang, W.G. Ma, S.Q. Lu, X.B. Xing, Detection of microRNA in clinical tumor samples by isothermal enzyme-free amplification and label-free graphene oxide-based SYBR Green I fluorescence platform, Biosens. Bioelectron., 65 (2015) 152–158. [27] X. Zhu, X.M. Zhou, D. Xing, Label-free detection of microRNA: two-step signal enhancement with a hairpin-probe-based graphene fluorescence switch and isothermal amplification, Chem. Eur. J., 19 (2013) 5487–5494. [28] F. Li, J.J. Li, Y. Feng, L.M. Yang, Z.F. Du, Electrochemical behavior of graphene doped carbon paste electrode and its application for sensitive determination of ascorbic acid, Sensors Actuators B: Chem., 157 (2011) 110–114. [29] L. Lin, Y. Liu, X. Zhao, J.H. Li, Sensitive and rapid screening of T4 polynucleotide kinase activity and inhibition based on coupled exonuclease reaction and graphene oxide platform, Anal. Chem., 83 (2011) 8396–8402. [30] K.S. Novoselov, A.K. Geim, S.V. Morozov, D. Jiang, Y. Zhang, S.V. Dubonos, et al., Electric field effect in atomically thin carbon films, Science, 306 (2004) 666–669. [31] H.F. Dong, J. Zhang, H.X. Ju, H.T. Lu, S.Y. Wang, S. Jin, et al., Highly sensitive multiple microRNA detection based on fluorescence quenching of graphene oxide and isothermal strand-displacement polymerase reaction, Anal. Chem., 84 (2012) 4587–4593. [32] F. Li, Y. Feng, C. Zhao, P. Li, B. Tang, A sensitive graphene oxide-DNA based sensing platform for fluorescence "turn-on" detection of bleomycin, Chem. Commun., 48 (2012) 127–129. [33] H.Y. Liu, L. Li, Q. Wang, L.L. Duan, B. Tang, Graphene fluorescence switch-based cooperative amplification: a sensitive and accurate method to detection microRNA, Anal. Chem., 86 (2014) 5487–5493. [34] L. Qiu, H. Zhou, W.P. Zhu, L.P. Qiu, J.H. Jiang, G.L. Shen, et al., A novel label-free fluorescence aptamerbased sensor method for cocaine detection based on isothermal circular strand-displacement amplification and graphene oxide absorption, New J. Chem., 37 (2013) 3998–4003.

12

[35] X.Z. Wang, A.W. Jiang, T. Hou, H.Y. Li, F. Li, Enzyme-free and label-free fluorescence aptasensing strategy for highly sensitive detection of protein based on target-triggered hybridization chain reaction amplification, Biosens. Bioelectron., 70 (2015) 324–329. [36] Q.S. Chen, W.L. Wei, J.M. Lin, Homogeneous detection of concanavalin A using pyrene-conjugated maltose assembled graphene based on fluorescence resonance energy transfer, Biosens. Bioelectron., 26 (2011) 4497–4502. [37] R.C. Huang, W.J. Chiu, Y.J. Li, C.C. Huang, Detection of microRNA in tumor cells using exonuclease III and graphene oxide-regulated signal amplification, ACS Appl. Mater. Interfaces, 6 (2014) 21780–21787. [38] Y.Q. Tu, W. Li, P. Wu, H. Zhang, C.X. Cai, Fluorescence quenching of graphene oxide integrating with the site-specific cleavage of the endonuclease for sensitive and selective microRNA detection, Anal. Chem., 85 (2013) 2536–2542. [39] S.R. Ryoo, J. Lee, J. Yeo, H.K. Na, Y.K. Kim, H. Jang, et al., Quantitative and multiplexed microRNA sensing in living cells based on peptide nucleic acid and nano graphene oxide (PANGO), ACS Nano, 7 (2013) 5882–5891. [40] L. Cui, X.Y. Lin, N.h. Lin, Y.L. Song, Z. Zhu, X. Chen, et al., Graphene oxide-protected DNA probes for multiplex microRNA analysis in complex biological samples based on a cyclic enzymatic amplification method, Chem. Commun., 48 (2012) 194–196. [41] H. Zipper, H. Brunner, J. Bernhagen, F. Vitzthum, Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications, Nucleic Acids Res., 32 (2004) e103.

13

Table 1. Sequences of the oligonucleotides used in the experiments Figure Captions Scheme 1. The principle of the label-free fluorescence strategy based on isothermal exponential amplification and graphene oxide absorption for miRNA assay. Fig. 1. (A) Fluorescence emission spectra of SG under different conditions: (a) SG; (b) SG + GO; (c) SG + GO + miRNA21; (d) SG + GO + ssDNA1; (e) SG + GO + HP; (f) SG + GO + dsDNA (hybridization product of ssDNA1 and ssDNA2). (B) The fluorescence intensity of SG at 520 nm in the presence of (a) miRNA21 + RNase + ssDNA2; (b) miRNA21 + GO + RNase + ssDNA2; (c) miRNA21 + ribonuclease inhibitor + RNase + ssDNA2; (d) miRNA21 + GO + ribonuclease inhibitor + RNase + ssDNA2. (C) Fluorescence emission spectra of SG in the presence of (a) HP + GO; (b) HP + GO + Primer2 + KF polymerase + dNTP; (c) HP + GO + Primer2 + KF polymerase + dNTP + Nb.BtsI; (d) HP + GO + Primer2 + KF polymerase + dNTP + Nb.BtsI + miRNA21. Fig. 2. (A) The relationship between the relative fluorescence intensity and the length of the primer. The fluorescence intensity versus (B) the concentration of HP and (C) the amount of KF polymerase. The error bars represent the standard deviation of three measurements. Fig. 3. Fluorescence emission spectra of the biosensing system upon the addition of miRNA21 with different concentrations: (a) 0 (control), (b) 10 fM, (c) 50 fM, (d) 100 fM, (e) 500 fM, (f) 1 pM and (g) 10 pM. Inset: the linear relationship between the fluorescence intensity change (F-F0) and the logarithm of miRNA21 concentration ranging from 10 fM to 10 pM. The error bars represent the standard deviation of three measurements. Fig. 4. Comparison of the fluorescence intensity of the biosensing platform at 520 nm in the presence of miRNA21, miRNA210 and miRNA214, respectively, in which “Control” indicates the condition in the absence of miRNAs. The concentrations of miRNA21, miRNA210 and miRNA214 were all 1 pM. The error bars represent the standard deviation of three measurements.

14

Highlights Ÿ

We have developed a label-free fluorescence strategy for sensitive and selective miRNA detection.

Ÿ

This strategy combines isothermal exponential amplification and the unique features of graphene oxide (GO) and SYBR Green I (SG).

Ÿ

GO exhibits multiple functions of protecting miRNA from enzymatic digestion, selectively adsorbing single-stranded DNA, and quenching the fluorescence of free SG.

Ÿ

This method avoids expensive and tedious labeling process and exhibits additional advantages of simplicity and low cost.

15

Table 1. Sequences of the oligonucleotides used in the experiments a Name

Sequence (from 5¢ to 3¢)

miRNA21

5¢-UAG CUU AUC AGA CUG AUG UUG A-3¢

miRNA210

5¢-CUG UGC GUG UGA CAG CGG CUG A-3¢

miRNA214

5¢-ACA GCA GGC ACA GAC AGG CAG U-3¢

HP

5¢-AAG GTA AAT CAA CAT CAG TCT GAT AAG CTA GCA GTG ACT CGA TGC TAG CTT ATC AG-phosphate-3¢

Primer1

5¢-AGC TAG-3¢

Primer2

5¢-AGC TAG C-3¢

Primer3

5¢-AGC TAG CA-3¢

Primer4

5¢-AGC TAG CAT-3¢

ssDNA1

5¢-TAG CTT ATC AGA CTG ATG TTG A-3¢

ssDNA2

5¢-TCA ACA TCA GTC TGA TAA GCT A-3¢

a

In HP, the boldface letters represent the sequences complementary to each other, and the underlined letters represent the recognition sequences of Nb.BtsI.

16

Graphical Abstract

Scheme 1

Fig. 1

Fig. 2

Fig. 3

Fig. 4