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Detection of microRNA : a point-of-care testing method based on a pH-responsive and highly efficient isothermal amplification Chang Feng, Xiaoxia Mao, Hai Shi, Bing Bo, Xiaoxia Chen, Tianshu Chen, Xiaoli Zhu, and Genxi Li Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 29 May 2017 Downloaded from http://pubs.acs.org on May 29, 2017

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Analytical Chemistry

Detection of microRNA: a point-of-care testing method based on a pH-responsive and highly efficient isothermal amplification Chang Feng,†,‡ Xiaoxia Mao,‡ Hai Shi,† Bing Bo,§ Xiaoxia Chen,‡ Tianshu Chen,‡ Xiaoli Zhu,*,‡ and Genxi Li*,†,‡ †

State Key Laboratory of Pharmaceutical Biotechnology and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Biochemistry, Nanjing University, Nanjing 210093, P. R. China. Tel.: +86-25-83593596, Fax: +86-2583592510, E-mail: [email protected] ‡ Center for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University, Shanghai 200444, P. R. China. Tel: +86-21-66138132, E-mail: [email protected] § Department of Medical Oncology, Shanghai Pulmonary Hospital Tongji University School of Medicine, Shanghai 200433, P. R. China. ABSTRACT: Laborious and costly detection of miRNAs has brought challenges to its practical applications, especially for home health care, rigorous military medicine and the third world. In this work, we present a pH-responsive miRNA amplification method, which allows the detection of miRNA just using a pH test paper. The operation is easy and no other costly instrument is involved, making the method very friendly. In our strategy, a highly-efficient isothermal amplification of miRNA is achieved using an improved netlike rolling circle amplification (NRCA) technique. Large amounts of H+ can be produced as a by-product during the amplification to induce significant changes of pH, which can be monitored directly using a pH test paper or pH-sensitive indicators. The degree of color changes depends on the amount of miRNA, making it possible for quantitative analysis. As an example, the method is successfully applied to quantify a miRNA (miR-21) in cancer cells. The results agree well with that from the prevalent qRT-PCR analysis. It is the first time that a paper-based point-of-care testing (POCT) is developed for the detection of miRNAs, which might promote the popularization of miRNAs working as biomarkers for diagnostic purposes.

MicroRNAs are an emerging class of short (20 ~ 23 nucleotides), non-coding RNAs that cause destabilization or posttranscriptional repression of target mRNAs. 1-2 They are involved in various biological processes, including the development and homeostasis of almost all the living organisms. Recent researches demonstrate that the aberrant expression of miRNAs is associated with many human carcinomas, such as lung cancer, hepatocellular carcinoma, breast cancer and gastric carcinoma, suggesting the bright prospect of miRNA to work as valuable biomarkers for early diagnosis of cancers. 3-8 Thus, the development of simple, specific and sensitive methods for the detection of miRNA is urgently needed. Currently, though qRT-PCR is considered a gold standard for miRNA analysis, it is still subjected to some drawbacks in practical applications e.g. the complex design of primers and the need of costly instrument. Given this, various strategies have been developed to improve the performance and adaptability of miRNA detection in recent years, 9 e.g. nanoparticlebased assay, 10 surface plasmon resonance-based assay 11 and enzyme catalytic amplification-based electrochemical assay.12 Nevertheless, in view of the lack of resources in some harsh circumstances like home health care, rigorous military medicine and the third world, there is still no proper method to realize the detection of miRNA with favorable cost-efficiency

and conveniences. In recent decade, the advent of point-ofcare diagnostic techniques provides a way for the health care in the abovementioned harsh circumstances. It is a rosy scenario to connect the analysis of miRNA with POCT. But, it is still a pity that the POCT has not covered the detection of miRNA either in theory or in practice yet.

EXPERIMENTAL SECTION Materials. All oligonucleotides used in this research (Table S1) were synthesized by TaKaRa Biotechnology Co., Ltd. (Dalian, China) with HPLC purification. Deoxynucleotide solution mixture (dNTPs) was obtained from the TaKaRa Biotechnology. and 6 × loading buffer was purchased from Shanghai Generay Biotechnology Co., Ltd. Nb. BsrDI nicking enzyme, Bst DNA polymerase large fragment, Hot Start Taq DNA Polymerase, Nt. BsmAI nicking enzyme and their corresponding buffer were purchased from New England Biolabs Inc. 8-tube strip (0.1 mL) was purchased from Shanghai Sangon Biotechnology Co., Ltd. SYBR Green I was from Solarbio Technology Co., Ltd. All indicator dyes were obtained from Sigma-Aldrich. pH-Indicator Strips were purchased from Merck Millipore. Other reagents were all of analytical grade and used as received. All solutions were prepared with Milli-Q

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water (18.2 MΩ cm−1) from a Milli-Q purification system (Millipore Corp, Milford, MA) Isothermal amplification reactions were performed on a ThermoMixer C (Eppendorf China Ltd.). Gel electrophoresis was conducted using a Wide Mini-Sub Cell GT Cell and a Gel Doc XR Imaging System (Bio-Rad, US). Absorbance was measured using a SpectraMax M3 Multi-Mode Microplate Reader with SoftMax Pro software (Molecular Devices, Sunnyvale, CA, USA). Polymerase chain reaction was conducted using a Mastercycler personal (Eppendorf, Germany). The pH values were measured with a pH meter (Mettler Toledo Instruments Co. Ltd., Shanghai, PR China). NRCA-based assay. The NRCA reaction was conducted in a 50 µL reaction mixture containing 26 µM Tris buffer, different concentrations of miRNA-21, 200 nM Primer 1 and Primer 2, 100 nM circular probe, 400 µM dNTPs, 10 U Nb. BsrDI nicking enzyme, and 8 U Bst. DNA polymerase, initial pH was adjusted with 1 M KOH to ~8.5. The reaction was allowed to proceed at 65 °C for 1 h and terminate by deactivating the enzymes at 95 °C for 10 min. In the case of hyperbranched rolling cycle amplification (HRCA), Nb. BsrDI was removed from the reaction system, while other conditions were kept unchanged. As for RCA, Primer 2 and Nb. BsrDI were both removed. PCR. The PCR reaction was performed in a 50 µL reaction mixture containing 26 µM Tris buffer, 20 pM loop stem primer, 30 pM forward primer, 20 pM reverse primer, 200 µM dNTP, different concentration of template, 5 U Taq DNA polymerase, 1.5 mM MgCl2, initial pH was adjusted with 1 M KOH to ~8.5. Amplification conditions were as follows: extension at 72 °C for 15 min and Initial denaturation at 95 °C for 5 min; followed by 35 cycles, each at 95 °C for 30 s, at 52 °C for 30 s, and extension at 72 °C for 30 s, and ended with an extension at 72 °C for 5 min and quickly chilled to 12 °C on a Mastercycler personal (Eppendorf, Germany). SDA. SDA was performed in a solution containing 26 µM Tris buffer, different concentrations of miRNA-21 and 200 nM template, 400 µM dNTPs, 8 U Bst. DNA polymerase, 10 U Nt. BsmAI, initial pH was adjusted with 1 M KOH to ~8.5. The reactions were incubated at 65°C for 1 h. Gel electrophoresis analysis. Agarose gel electrophoresis was performed for the characterization of the products of Immuno-NRCA. 5 µL of the products of Immuno-NRCA with 1 µL 6 × loading buffer was loaded onto a 2% non-denaturing agarose gel. The electrophoresis experiments were carried out in 1 × Tris-acetate-EDTA (TAE) at 80 V for 30 min. Subsequently, the gel was stained with SYBR Green I for 30 min. The imaging of the gel was performed using a Gel Doc XR Imaging System. Absorbance detection. 2.5 mM of pH indicator (Cresol red, Neutral red, m-Cresol purple) were added into 50 µL of NRCA products after reaction, visually inspected for color change after removal from reaction temperature and scanned. Absorbance spectra were obtained using a SpectraMax M3 Multi-Mode Microplate Reader and the spectra were measured in the range from 300 nm to 700 nm. The corresponding absorbance of Cresol red, Neutral red and m-Cresol purple were recorded at 570 nm, 550 nm and 579 nm, respectively. Cell culture and RNA preparation. All cells obtained from the Institute of Biochemistry and Cell Biology (Chinese Academy of Science). Invasive breast ductal carcinoma

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(MCF-7) was cultured in RPMI supplemented with 10% fetal bovine serum (FBS). Human hepatocellular carcinoma (HepG2) and HeLa cell were cultured in DMEM supplemented with 10% FBS. All cells were maintained in a humidified incubator at 37 °C with 5% CO2 and 80% relative humidity. Total RNA of MCF-7, HepG2 and HeLa were extracted using TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol. Briefly, the sample was lysed and homogenized with TRIzol Reagent. Phase separation was followed by addition of chloroform and centrifugation. RNA in the aqueous phase was then recovered by precipitation with isopropyl alcohol. The RNA pellet was washed with 75% ethanol and finally redissolved in RNase-free water. The RNA quantity was determined by measuring optical density at 260 nm using the Biophotometer (Eppendorf, Germany).

RESULTS AND DISCUSSION Principle of the POCT method for the detection of miRNA. Herein, we attempt to develop a POCT method for the detection of miRNA. An isothermal amplification technique termed as netlike rolling circle amplification (NRCA) is borrowed to amply the signal as well as to abandon using of costly thermal-cycler. Figure 1A illustrates the amplification strategy, which can be also referred to our previous reports. 1314 A DNA polymerase extends the primer 1 using a circular probe as the template. This reaction is well known as the RCA. While a reverse primer 2 is further presented to bind with the extended RCA products, ramified extension and strand displacement proceed simultaneously in addition to the RCA reaction, resulting in the HRCA. In our NRCA system, a nicking enzyme is further involved based on HRCA to allow the cycle of three amplification processes (rolling cycle amplification, strand displacement and nicking reaction). Here the nicking enzyme scissors the HRCA products from specific sites into fragments, which can work as primers separated to launch new HRCA reactions, producing a much higher amplification efficiency. During the amplification, the released byproducts include a pyrophosphate moiety and a hydrogen ion (H+, Figure 1B), 15-16 the amount of which depends strongly on the amplification efficiency. Given the high enough amplification efficiency as well as right conditions, it is expected that the changes of pH induced by the production of H+ will be significant enough to be monitored just using a pH sensitive indicator or a pH test paper (Figure 1C). In this work, miR-21, which has been widely reported to overexpress in cancer cells, is adopted as a model target (Figure 1D). The successful implement of the above scheme would contribute to the low-cost diagnosis of cancers using miRNAs as biomarkers. Proof-of-concept. In order to make the results readable, single pH sensitive indicator is first adopted, the color changes of which can be quantitatively analyzed using UV-vis spectrometry. Three indicators including cresol red, neutral red and m-cresol purple, whose critical pH for color changes are all around the most sensitive neutral pH, are selected as candidates. Some experimental conditions that play key roles for the pH changes, including the initial pH of the amplification processes (Figure S1), the amplification time (Figure S2) and the buffer capacity of the buffer solution (Figure S3), are optimized. After that, qualitative results under the optimized conditions (initial pH: 8.5; amplification time: 60 min; concentration of Tris-HCl buffer: 26 µM) show that apparent color

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Analytical Chemistry the concentration, it is adopted for quantitation. A log-linear relationship between the absorbance and the target miR-21 in a range from 20 fM to 20 nM (R2 = 0.99) can be obtained. The detection limit is calculated to be 9.3 fM (LOD = 3SD / k, LOD: detection limit, SD: the standard deviation of blank sample, k: the slope of the fitting curve), a value rivaling some other methods. 17-19 In the case of neutral red and m-cresol purple, the increased peak at 550 nm and the decreased peak at 579 nm are selected respectively for quantitation. The detection limits are 15.80 fM and 35.07 fM, respectively. To further investigate the specificity of the proposed detection method, we performed experiments using single-base-mismatched miR-21 (SM miR-21), three-base-mismatched miR-21 (TM miR-21), miR-210 and miR-203 as negative controls. As is shown in Figure S4, the colorimetric signals of SM miR-21 are slightly different from the miR-21, while TM miR-21, miR-210 and miR-203 exhibit much significant differences, which can be discriminated easily by naked eyes. Therefore, our proposed method shows a good selectivity when challenged with other miRNAs.

changes can be observed after the amplification of 200 nM miR-21 using any of the three indicators (Figure 2). In control experiments that the polymerase is absent, there is no color change, suggesting the expected amplification-induced color changes.

Figure 1. Schematic presentation of the principle of pHresponsive NRCA-based colorimetric assay of miRNA. (A) The brief principle of NRCA. (B) The equation of the reaction catalyzed by Bst. DNA polymerase. (C) Color changes of the adopted pH-responsive indicators under different pH. (D) Prospect for cancer diagnosis through detecting miRNA with pH test papers. Figure 3. Quantitative analysis of miR-21 using the pHresponsive NRCA-based colorimetric strategy with pH indicators (from left to right: cresol red, neutral red and m-cresol purple). From top to bottom: appearance of the color changes with the increasing of the concentration of miR-21, UV-vis absorption spectra and the background-subtracted absorbance at 570 nm, 550 nm and 579 nm respectively for each indicator as a function of the miR-21 concentration (the insets show the linear calibration plots). Studies of the stability of the colorimetric signal outputs. In view of the harsh circumstances of nonlaboratory assays, favorable stability of the output signal, i.e. the final color appearance, is required. In experiments, the color stability is studied under different static duration, temperature and lighting conditions. From Figure S5 and S6, it can be concluded that the colorimetric results using any of the three indicators can be retained under a large range of temperature from 0 to 65 °C, and under either sunlight or UV light irradiation. The results suggest the favorable stability of the color appearance, which is important for the untrained users from different regions and in different circumstances to read and analyze. As for the static duration however, the color stability differs for miR-21 negative and positive samples (Figure S7). In the case of a miR-21 negative sample, amplification is not launched, resulting in the initial alkaline pH unchanged at first but sensitive to the slow-dissolving of CO2 from the at-

Figure 2. Detection of 200 nM miR-21 using the pHresponsive NRCA-based colorimetric strategy with pH indicator dyes (from left to right: cresol red, neutral red and m-cresol purple) using naked-eye observation and UV-vis spectroscopy. Detection of miR-21 using the POCT method. Detection of the target miR-21 with different concentrations using the color changes of the indicators is then performed. As is shown in Figure 3, with the increasing of the miR-21 concentration, the color changes after amplification become more and more apparent. UV-vis spectra show accurate quantitative absorption data of the changes. For cresol red, the peak at 570 nm decreases sharply, and a new peak at 430 nm emerges. Considering the better relevance of the peak at 570 nm with

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and HeLa (immortal cell line). They are regarded as common breast, liver and cervical cancer cell models, respectively. Considering the better performance of cresol red in the detection limit, here this indicator together with pH test papers is adopted. As is shown in Figure 5, the total amount of miR-21 in 1,000 MCF-7 cells presents an easily distinguished color change for either cresol red or pH test paper. While in the case of HepG2 and HeLa, the critical cell numbers are 2,000 and 5,000, respectively, suggesting a gradient of miR-21 amount: MCF-7 > HepG2 > HeLa. Corresponding UV-vis spectroscopic results are shown in Figure S13. According to the calibration curve shown in Figure 3, the amount of miR-21 in total RNA of each cell line is found to be 1.16, 0.48 and 0.41 amol/ng for MCF-7, HepG2 and HeLa, respectively. The results have also been confirmed by qRT-PCR (Figure 6), whose calibration curve can be found in Figure S14.

mosphere. After about 5 min, the colors especially for cresol red and m-cresol purple will change owing to the H2CO3induced acidification. But for a miR-21 positive sample, the amplification has resulted in an acidic pH, which is not sensitive to the dissolved H2CO3 anymore. The above speculation is further confirmed by purging the solutions with nitrogen and keeping them in a nitrogenous atmosphere throughout the observation to avoid the dissolving of CO2 (Figure S7). As a practical alternative, favorable performance can be also obtained by just sealing the test tubes (Figure S7). Thus, the color output can keep stable in air for over 1 hour.

Figure 4. Comparison of different isothermal amplification techniques (HRCA, SDA and NRCA) for the detection of miR-21 using cresol red as the indicator. From top to bottom: appearance of the color changes with the increasing of the concentration of miR-21, corresponding UV-vis absorption spectra, the linear calibration plots of the absorbance at 570 nm vs. the concentration of miR-21. Comparison of the performance of different isothermal amplification techniques. Because some other polymerase-based isothermal amplification techniques may also support similar pH-dependent colorimetric detection of miRNA, a systematic comparison between the adopted NRCA technique and some conventional isothermal amplification techniques is conducted. Also considering the physiological concentration of miRNA is mainly in a level from fM to pM, 20-21 the performance for the detection of miR-21 in this range is compared. The results using cresol red as the indicator are shown in Figure 4, while the others are shown in the supporting information (Figure S8~S10). As is shown, though a similar linear detection range can be obtained by using different amplification techniques, the critical concentration that can be detect by naked eyes differs from each other (10 pM, 1 pM and 100 fM for HRCA, SDA, and NRCA, respectively). That is, NRCA has the best performance for macroscopic observation during these isothermal amplification techniques. The conclusion can be also supported by using other indicators (Figure S8~S10). Quantification of miR-21 in cancerous cell lines. After confirming that the pH-responsive colorimetric detection strategy is barely affected by the complexity of physiological samples (Figure S11), we detect miR-21 in spiked serum samples and cancer cells. For serum samples, the results for the detection of spiked miR-21 coincide well with that in buffer solutions (Figure S12). While for cancer cells, three cell lines are adopted, including MCF-7 (invasive breast ductal carcinoma cell line), HepG2 (human hepatocarcinoma cell line)

Figure 5. Detection of miR-21 in total RNA (5 ng per 1,000 cells) from different cell lines using pH-responsive NRCAbased colorimetric assay with cresol red or pH test paper.

Figure 6. Comparison of the pH-responsive NRCA-based colorimetric assay and qRT-PCR assay for the determination of miR-21 contents in MCF-7, HepG2 and HeLa cells.

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Analytical Chemistry Present Addresses

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† State Key Laboratory of Pharmaceutical Biotechnology and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Biochemistry, Nanjing University, Nanjing 210093, P. R. China.

CONCLUSION In summary, we report here a paper-based POCT method for the detection of miRNAs, a kind of biomarker holding the great potential for diagnosis. A highly-efficient isothermal amplification is adopted to abandon the usage of costly thermal-cycler. Owing to the by-production of H+, a significant change of the system pH from alkalinity to acidity is excitingly observed during the miRNA amplification after extensive and elaborate trials. Thus, we are able to detect miRNA using pH-sensitive indicators or just a pH test paper. The method is welcome especially for home health care and those harsh circumstances. Detection of miR-21, a tumor-associated miRNA, in both spiked serum samples and cells is successfully achieved. For the determination of miR-21 positive or negative sample, a pH test paper may help; and if accurate quantitation is required, colorimetric assay of the pH-sensitive indicator is preferred, which is also applicable to professional laboratory for quick quantification. This method that enables the detection of miRNA with favorable cost-efficiency and conveniences might promote the popularization of miRNAs working as biomarkers for diagnostic purposes.

‡ Center for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University, Shanghai 200444, P. R. China. §Department of Medical Oncology, Shanghai Pulmonary Hospital Tongji University School of Medicine, Shanghai 200433, P. R. China.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grant Nos. 21575088, 21235003) and the Natural Science Foundation of Shanghai (14ZR1416500).

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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Sequences of oligonucleotides; Investigation of the optimal initial pH of the pH-responsive NRCA-based colorimetric assay using three pH indicators; Investigation of the optimal amplification time of the pH-responsive NRCA-based colorimetric assay; Investigation of the optimal concentration of Tris-HCl buffer; Specificity of the NRCA-based colorimetric strategy for the detection of miRNA-21; Thermal stability of the color outputs after assay; Irradiation stability of the color outputs after assay; Investigation of the stability of the color outputs as a function of static duration; Comparison of different isothermal amplification techniques (HRCA, SDA and NRCA) for the detection of 200 nM miR-21; Comparison of different isothermal amplification techniques (HRCA, SDA and NRCA) for the detection of miR-21 using neutral red as the indicator; Comparison of different isothermal amplification techniques (HRCA, SDA and NRCA) for the detection of miR-21 using m-cresol purple as the indicator; Detection of spiked miR-21 (200 nM) in diluted serum samples using pHresponsive NRCA-based colorimetric assay; Detection of different concentrations of spiked miR-21 in diluted serum samples using the pH-responsive NRCA-based colorimetric assay; UV-vis absorption spectra using cresol red as the indicator for the detection of miR-21 in different cell lines by using different cell numbers; Calibration curve for qRT-PCR assay of miR-21 (PDF)

AUTHOR INFORMATION Corresponding Author * E-mail addresses: [email protected] * E-mail addresses: [email protected].

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