- Email: [email protected]
Ultrasensitive detection of microRNA-21 based on electrophoresis assisted cascade chemiluminescence signal amplification for the identification of cancer cells
Talanta xxx (xxxx) xxxx
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Ultrasensitive detection of microRNA-21 based on electrophoresis assisted cascade chemiluminescence signal amplification for the identification of cancer cells Caimei Hea, Shengyu Chena, Jingjin Zhaoa,b,∗, Jianniao Tiana, Shulin Zhaoa a b
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University, Guilin, 541004, China Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection, Ministry of Education, Guangxi Normal University, Guilin, 541004, China
ARTICLE INFO
ABSTRACT
Keywords: miRNA detection Microchip electrophoresis Signal amplification Chemiluminescence
Rapid and accurate detection of microRNA content in cells is of great significance. Here, an ultrasensitive microchip electrophoresis (MCE) method based on cascade chemiluminescence (CL) signal amplification was developed for the detection of microRNA-21 in cells. In this method, horseradish peroxidase labeled DNA was used as a signal probe, which could induce CL signal by the reaction of luminol and H2O2. Combining with two cyclic enzyme digestion reactions by T7 exonuclease, a large number of signal probes were degraded. By using MCE-CL as a separation and detection platform, an amplified CL signal peak was achieved. The developed MCE-CL method can detect miR-21 at a concentration as low as 1.0 × 10−15 M, which was enhanced by six orders of magnitude compared with those of conventional MCE-CL assay. This method has been applied for the detection of microRNA-21 in cell lysate, which show that there were significant differences of miR-21 among different types of cells, and the content in cancer cells was much higher than that in normal cells, which can be used for the identification of cancer cells. Therefore, the proposed method held great application potential in early diagnosis of tumor and biomedical research.
1. Introduction MicroRNA-21 (miR-21) is a small RNA molecule with high expression in tumor. It has been found that human miR-21 is located in the 17q23.2 region of chromosome, and is related to many diseases [1]. The miR-21 is a carcinogenic miRNA, which is the only high expression miRNA found in multiple solid and non-solid tumors [2,3]. As an important miRNA with tumor gene properties, miR-21 can regulate the growth of breast cancer cells [4]. Its gene cluster can promote many kinds of tumors, and it has similar oncogene function in tumorigenesis and development. It also involved in tumor angiogenesis, and closely related to tumor cell proliferation, invasion and drug resistance [5,6]. In recent years, miR-21 may become a new biomarker for early diagnosis and gene therapy with the further study of its function [7,8]. Therefore, accurate detection of miR-21 in cells is of great significance for early diagnosis of related diseases and the discovery of new anticancer drugs. The content of miR-21 in human body is very low, raising the difficulties to detect its concentration with traditional methods. Moreover,
the most urgently need is to detect miRNA accurately in a complex biological matrix. A series of analytical methods have been developed for the determination of miR-21 by using various techniques to improve the sensitivity and selectivity. For example, Shi et al. reported an enzyme-free target recycling signal amplification electrochemical sensor [9]. Zhen et al. proposed an enzyme-free DNA circuit-assisted graphene oxide enhanced fluorescence anisotropy assay [10]. Liu et al. developed an inductively coupled plasma mass spectrometry method for the determination of miR-21 in human plasma [11]. Park et al. proposed a colorimetric sensing method on the basis of the plasmonic coupling effect [12]. Liu et al. developed an electrochemical analysis method based on the layer-by-layer assembly of oxidized single-walled carbon nanotubes and nanodiamonds on the electrode [13], and Shuai et al. proposed a nanocrystalline gold/hollow molybdenum disulfide micro cube biosensor [14]. However, most of these methods are cumbersome, require a large volume of sample or with high background. To this end, the development of simple, rapid, low-consumption and high-sensitivity analytical methods for miR-21 detection is still a subject of high attention.
∗ Corresponding author. State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University, Guilin, 541004, China. E-mail address: [email protected] (J. Zhao).
https://doi.org/10.1016/j.talanta.2019.120505 Received 14 June 2019; Received in revised form 21 October 2019; Accepted 24 October 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Caimei He, et al., Talanta, https://doi.org/10.1016/j.talanta.2019.120505
Talanta xxx (xxxx) xxxx
C. He, et al.
As a new separation and analysis platform, the microchip electrophoresis (MCE) has the advantages of low sample and reagent consumption, fast analytical speed, high throughput and automation [15–18]. In recent years, MCE technique has been applied in biochemical [19–21], single cell [22,23] and clinical analysis [24–26]. However, the sensitivity of conventional MCE analysis is relatively low because of its extremely small sample size, which limits its application in the detection of trace substances in organisms. To overcome this drawback, a variety of methods have been proposed to improve the sensitivity of MCE technology. For example, kawai et al. proposed a large-volume sample stacking method, and the sensitivity was increased 2200–2900 times [27]. Wang et al. proposed a multiple-concentration approach combining chitosan sweeping, field-amplified sample stacking, and reversed-field stacking, with the sensitivity increasing 6000 times [28]. Tang's team put forward the strategy of nucleic acid adaptation and enzyme-assisted signal amplification, which made the detection limit of microchip platform reach 1.9 × 10−11 M [29]. Although the sensitivity has been improved a lot by these methods, it is still a challenge to realize the accurate determination of miRNA on the MCE platform due to its low content in organisms. Chemiluminescence (CL) is a powerful and widely used analytical technique with merits of low background, high sensitivity and wide linear range. Coupling with CL detection, MCE-CL method has attracted great attention for its good performance in analysis [30]. Some of them have been successfully used for detection of DNA fragments, enzymes and proteins, which indicates that CL is a powerful and promising technique for MCE assay. The present work integrates the advantages of MCE technology and CL reaction of between luminol and hydrogen peroxide (H2O2), combining with two cyclic enzyme digestion reaction of DNA probes by T7 exonuclease (T7exo). An electrophoresis assisted cascade CL signal amplification method was developed for ultrasensitive detection of miRNA-21. The limit of detection of this MCE-CL platform reached 1.0 × 10−15 M. This strategy has been applied for the detection of miRNA-21 in cell lysate, and realize the recognition of miR-21 in both cancer cells and normal cells.
(Shanghai Precision Scientific Instrument Co. Ltd), SH-III water circulating vacuum pump (Zhengzhou Great Wall Scientific Industrial Co. Ltd), and FS-150N ultrasonic instrument (Shanghai shengxi ultrasonic instrument Co., Ltd). 2.2. Cascade amplification reaction The single-stranded DNA probe H1 and H2 were diluted to 2 μM with Tris-HCl buffer respectively, and then heated at 95 °C for 5 min, followed by cooling to room temperature naturally. After that, 4 μL of H1 and H2, 2 μL of different concentration of miRNA, 1.5 μL of 10 × NEBbuffer 4 and 6.5 μL of Tris-HCl buffer were added to a 100 μL centrifuge tube, respectively. Then 4 μL of 1.6 × 10−7 M HRP-DNA probe and 3 μL of 10 U/μL enzyme T7exo were added in the above solution. After mixed and incubated at 37 °C for 120 min in a water bath, 29 μL of Tris-HCl buffer was added to a volume of 50 μL before MCE-CL analysis. 2.3. Microchip electrophoresis analysis Before new chip was used, the channels were flushed with 10 mM SDS and methanol solution for 5 min each, then rinsed with 1 M hydrochloric acid for 10 min, 0.1 M sodium hydroxide for 30 min. Between two sample runs, the channel was sequentially activated with 0.1 M NaOH solution for 10 min, rinsed with water for 5 min. Subsequently, each reservoir was filled with electrophoresis buffer, and filled chip channels using a vacuum pump. Then, the sample solution and CL buffer solution were used to replace the solution in the S and the R reservoirs, respectively. The sample solution was injected into the separation channel in pinched mode by applying 500 V, 250 V and 400 V voltages to S, B and BW reservoirs. SW reservoir grounding and R reservoir was suspended. After 20 s, the voltage of each reservoir was switched, 2400 V was applied to the B reservoir as the separation voltage, 1500 V voltage was applied to the S and SW as the pull back voltage, while the BW reservoir was grounded, and 500 V voltage was applied in the R reservoir. Under the action of electroosmotic flow, the analytes migrate to the Y-type intersection at different rates in the separation channel, and mixed with the oxidizer to generate CL signal, which were collected by the photomultiplier tube (PMT).
2. Experimental 2.1. Reagents and apparatus
2.4. Preparation of cells lysate
The nucleic acid molecules used in this study were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China), which are shown in Table S1. Luminol was purchased from Fluka Co., Ltd. (Switzerland), and the P-piphenol (PIP) was obtained from Shanghai Bangcheng Chemical Co., Ltd. (China). H2O2, sodium dodecyl sulfate (SDS) and borax were purchased from Shanghai Chemical Reagent Co., Ltd (Shanghai, China). Trimethylamino methane (Tris) was achieved from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). T7 exonuclease (T7exo) and 10 × NEB buffer 4 were purchased from New England Biolabs Inc. (Beverly, MA, USA). Other chemicals used in this work were of analytical grade. Ultrapure water (18.2 MΩ) was used throughout the experiments. The solutions used in this work include: DNA storage solution (2.0 × 10−5 M in 1.0 × 10−2 M Tirs-EDTA buffer, pH = 8); TirsHCl buffer (1.0 × 10−2 M Tirs-HCl, 20 mM KCl, 0.2 M NaCl, pH = 7.4); electrophoresis buffer (25 mM borate, 15 mM SDS, 1.4 mM luminol, pH = 9.4) and CL reaction buffer (35 mM NaHCO3, 130 mM H2O2, pH = 10.5). MCE analysis was carried out on a self-assembled MCE-CL detection system by our laboratory [31]. The versatile programmable eight-pathelectrode power supply was developed and provided by Shandong Normal University [32]. Chromatography data system was purchased by Zhejiang University Zhida Information liability Co., Ltd. The chip used in this work is double T-type glass microchip with broadened Ytype CL detection cell, which was fabricated by Dalian Tuowei Technology Co., Ltd. The dimensions of the microchip was shown in Fig. S1. Other instruments used in this study include PHSJ-4A pH meter
The cells used in the experiment are human breast cancer cells (MCF-7), bladder cancer cells (T24) and normal human bladder cells (SV-HUC-1). The cell lines were cultured in a DMEM culture medium containing 10% fetal bovine serum, 10 U mL−1 penicillin, 10 g mL−1 streptomycin, and incubated at 37 °C for 24 h in a 5% CO2 atmosphere. After digesting with trypsin, the culture medium was removed by centrifugation, and the cells were dispersed in the PBS buffer. This procedure was repeated three times. Then, the cells were resuspended in 10 mM Tris-HCl buffer (pH = 7.4) with a cell density of about 1.0 × 104 cells mL−1. A 3 mL volume of above cell suspension was added into the centrifuge tube, and centrifuging for 5 min at a speed of 1000 r/min. Then, the PBS solution was abandoned, 200 μL Tris-HCl buffer solutions were used to suspend the cells, and transfer into a 1.5 mL centrifuge tube. The centrifugal tube was put in ice water bath on the ultrasonic instrument, with 80 W ultrasonic power ultrasonic broken for 20 min. After ultrasonic fragmentation, ultrafiltration membranes with a molecular weight cutoff of 10 kDa were used to remove large molecular weight proteins and extract the filtrate was retained for analysis. 2.5. Gel electrophoresis Agarose gel electrophoresis tests were carried out to verify the hybridization of miR-21, H1 and H2, and cleavage was induced by T7exo. 2
Talanta xxx (xxxx) xxxx
C. He, et al.
Fig. 1. Schematic diagram for MCE method based on separation assisted cascade CL signal amplification. Fig. 2. (A) The electrophoretograms for feasibility verification. (a) 0.16 μM HRPDNA, (b) 0.16 μM HRP-DNA+100 fM miR21, (c) 0.16 μM HRP-DNA+1 pM miR-21, (d) 0.16 μM HRP-DNA+10 pM miR-21. Peak identification: 1. HRP-DNA, 2. HRPlabeled monucleotide fragment. (B) Agarose gel electrophoresis characterization. Lane 1: miR-21.2: HRP-DNA. 3: H1/H2. 4: HRPDNA + H1/H2. 5: H1/H2+miR-21.6: H1/ H2+HRP-DNA + T7exo. 7: H1/H2+miR21+HRP-DNA + T7exo. Concentrations of HRP-DNA, H1/H2, miR-21 and T7exo are 3 μM, 3 μM, 2 μM and 40 U, respectively.
First, 4 g agarose was mixed with 100 mL of 0.5 × TBE and the mixture was heated by microwave for 3 min to produce agarose gel. Before electrophoresis, SYBR Green I, SYBR Green II was added to singlestranded DNA, double-stranded DNA samples and incubated for 10 min. After 4 μL of loding buffer was added, 60 min electrophoresis was performed at 100 V constant voltage. The electrophoresis results were observed using the Omega-16ic imaging system and photographed for analysis.
with the signal probe HRP-DNA to form a H2/HRP-DNA duplex, which could also be degraded by T7exo to release H2. The released H2 hybridized with another HRP-DNA probe to initiate the next cycle. As a result, amounts of HRP-labeled single-stranded DNA fragments were produced during the degradation process by T7exo. When CL signal was made through the HRP and luminol-H2O2 reaction, a cascade signal amplification could be achieved. The HRP-labeled DNA fragments and undigested HRP-DNA probes were separated and two peaks were present in the electropherogram after the MCE separation. The peak height of the HRP-labeled single-stranded DNA fragments was used for the quantification of miR-21.
3. Results and discussion 3.1. Method design and principle
3.2. Feasibility study of the method
The working principle of the method is shown in Fig. 1. First, DNA probe H1 hybridized with H2 to form a partially complementary duplex H1/H2. The prominent 5’ ends of DNA probes could prevent degradation by T7exo in the absence of target miR-21. T7exo is a doublestranded DNA specific exonuclease which can catalyze the removal of nucleotides from linear or nicked double-stranded DNA in the 5′ to 3′ direction. So, when miR-21 hybridized with H1 probe, T7exo recognized and degraded the duplex H1/H2 along the 5′→3′ direction of DNA sequence, releasing the target (miR-21) and H2 probe. Then, the released miR-21 continued to hybridize with remaining H1/H2 and triggered N cycles digestion of H1 probe by T7exo, resulting in the release of large numbers of H2 probes. The displaced free H2 hybridized
In order to verify the feasibility of the method, the effects of different miR-21 concentrations on the peak intensity of the HRP-labeled DNA fragments was investigated, and the experimental results are shown in Fig. 2A. In the absence of miR-21, the resulting electrophoresis curve a showed only one electrophoresis peak in the MCE-CL test. When a trace of miR-21 was added, the enzyme-assisted cascade amplification reaction was initiated and HRP-labeled DNA fragments were produced. The positions of the two peaks are different due to the difference of charge-to-mass ratio between the HRP-labeled DNA fragment and the HRP-DNA probe. The charge-to-mass ratio of the former is higher than that of the latter, so that the peak of HRP-labeled DNA 3
Talanta xxx (xxxx) xxxx
C. He, et al.
Fig. 3. (A) Electrophoretograms for detection of miR-21 in the concentration range of 5.0 fM~10 nM. (B) Dynamic curve of miR-21 detection (inset is linear curve of miR-21 detection). CL reaction condition: 120 mM H2O2; 1.2 mM luminol; 1.0 mM PIP; CL buffer pH = 10.5; 0.16 μM HRP-DNA; 30 U T7exo. MCE separation condition: 20 mM borax; 15 mM SDS; electrophoresis buffer pH = 10; voltage 2400 V. Peak identification: 1. HRP-DNA, 2. HRP-labeled nucleotide fragments.
fragment is firstly presented in curve b, c and d. With the increase of the amount of miR-21, the amount of the HRP-labeled DNA fragment was also increased, and the intensity of peak 2 gradually enhanced, while the intensity of peak 1 for the HRP-DNA probe decreased. The experimental results indicated that the proposed method is feasible for miR21 detection. Agarose gel electrophoresis tests were carried out on different systems to further verify the feasibility. As shown in Fig. 2B, two bands were observed in lane 4 which contained the mixture of H1/H2 and HRP-DNA, indicating that HRP-DNA could not hybridize with H2 in the absent of taget. When miR-21 was added with H1/H2, only one band in a backward position could be seen in lane 5, and a fully complementary double-stranded DNA was formed. Compare with lane 4, T7exo was added in lane 6, the two bands in the same position were still present because T7exo can not degrade the H1/H2 duplex without target. In the present of miR-21, the band of H1/H2 duplex was disappeared in lane 7, which means that H1 probe was degraded by T7exo. The above results prove the feasibility of the method again.
buffer, the concentration of SDS and borax in the electrophoresis buffer. The electrophoresis separation voltage in the range of 2300–2500 V was examined in Fig. S3A. When the voltage increases from 2300 V up to 2400 V, the resolution (Rs) of the sample increases gradually, and the Rs decreases when the voltage continues to increase, which may be due to the excessive Joule heat generated when the voltage is too large, and the bubbles are produced during the electrophoresis process. Therefore, the optimal separation voltage is selected as 2400 V. The effect of pH value of electrophoresis buffer solution in the range of 9.0–11.0 on the Rs are shown in Fig. S3B, and pH = 10 is chosen as the optimal pH value of electrophoresis buffer solution. SDS is an anionic surfactant which is commonly used in the separation of proteins and lipids. It can improve the electrophoretic separation. In our work, when the concentration of SDS was 15 mM, the Rs reaches the maximum value (Fig. S3C). The effect of borax concentration in the concentration range of 10–30 mM on the samples separation are shown in Fig. S3D The Rs reached the maximum value at 20 mM and then decrease gradually as the concentration of borax continues to increase, which is due to the excessive Joule heat caused by the excessive ion concentration of electrophoretic buffer solution.
3.3. Optimization of experimental conditions Several experimental conditions were investigated to achieve a better perfomance, including the concentrations of luminol, H2O2 and PIP, the pH value, T7exo dosage and reaction time. As shown in Figs. S2A and S2B, CL intensity reached the maximum when the concentrations of luminol and H2O2 are 1.2 mM and 120 mM, respectively. The sensitivity of the CL system can be improved by using CL sensitizers such as phenols. PIP was used as the sensitizer in our work, and the effect of PIP in the concentration range of 0.6–1.5 mM on the CL intensity was investigated. When the PIP concentration was 1.0 mM, the maximum CL intensity was obtained (Fig. S2C). Fig. S2D show the effect of the pH value of post-column buffer solution on the CL intensity, and pH = 10.5 was selected for the best pH value of the buffer solution. The effect of T7exo concentration in the range of 10–40 U on the CL intensity is shown in Fig. S2E. When the concentration of T7exo increased from 10 U to 30 U, the CL intensity of HRP-labeled monucleotide fragment increased gradually and then remained basically unchanged. The optimal concentration of T7exo in this study was 30 U. The effect of reaction time is presented in Fig. S2F, after the reaction time is longer than 140 min, the CL intensity almost keep constant. Therefore, the optimized reaction time is 140 min. The MCE separation conditions were also optimized, such us the electrophoresis separation voltage, the pH value of the electrophoresis
3.4. The assay performance for miR-21 detection Under the optimized experimental conditions, the analytical performances of this assay were investigated, and the experimental results are shown in Fig. 3. The peak height of the HRP-labeled DNA fragments raises gradually with the increase concentration of miR-21 (Fig. 3A, peak 2). As seen in Fig. 3B, it has a good linear relationship between peak signal and the logarithmic value of miR-21 concentration in the range from 5.0 fM to 10 nM. The linear equation is Y = 1.943X +11.664. Where Y represents the peak 2 height (mV), X represents the logarithmic value of miR-21 concentration, and the correlation coefficient R2 is 0.9967. The detection limit was calculated to be 1.0 fM with S/N = 3. 3.5. Study on the method specificity The miRNA is a kind of short-chain RNA with high sequence homology. In order to determine the target miRNA accurately, the specificity of the method must be investigated. Therefore, the effects of single base mismatch SM-21 and other miRNAs such as miR-15, miR-16 were investigated, the results are shown in Fig. 4. Compared with miR21, the CL intensity of other miRNAs are similar to that of the blank 4
Talanta xxx (xxxx) xxxx
C. He, et al.
4. Conclusions In summary, an ultrasensitive MCE method based on cascade CL signal amplification was developed for the detection of microRNA-21. As far as we know, this is the first cascade CL signal amplification method based on MCE-CL platform for the detection of miR-21. The advantages of this assay include simple operation, high degree of automation, short analysis time, low consumption of reagents and samples, very high sensitivity and good specificity. The detection limit for miR-21 was enhanced by six orders of magnitude compared to those of conventional MCE-CL method. This assay has been applied for the detection of miR-21 in cell lysate, and results show that there were significant differences of miR-21 among different types of cells. The content in cancer cells was much higher than that in normal cells, which can be used for the identification of cancer cells. Therefore, it has a wide application prospect in the early diagnosis of tumors and biomedical research. Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled.
Fig. 4. Study on the specificity of the method. The concentration of miR-21 is 6.0 pM. The concentrations of SM-21, miR-15, miR-16 and miR-141 are 600 pM, respectively. The experimental conditions are the same as in Fig. 3.
Table 1 The results for the detection of miR-21 in human cells. Samples
Founda (pM)
Added (pM)
Total found (pM)
RSD (%,N = 5)
Recovery (%)
MCF-7
0.139
T24
0.058
HCV-29
ND
0.250 0.500 0.750 0.250 0.500 0.750 0.250 0.500 0.750
0.412 0.642 0.884 0.315 0.552 0.822 0.260 0.504 0.764
2.7 1.6 1.1 2.8 1.7 1.6 2.6 2.0 1.6
109.2 100.6 99.3 102.8 98.8 101.8 104.0 100.8 101.8
a
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21765003), Natural Science Foundation of Guangxi Province (No. 2017GXNSFFA198014) and BAGUI Scholar Program. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2019.120505.
ND: No miR-21 was detected.
References
under the same experimental conditions, and the CL intensity of mismatch SM-21 is a quarter of that with target miR-21. The aboved results indicated that proposed method has a high specificity.
[1] T. Thum, C. Gross, J. Fiedler, T. Fischer, S. Kissler, M. Bussen, P. Galuppo, S. Just, W. Rottbauer, S. Frantz, M. Castoldi, J. Soutschek, V. Koteliansky, A. Rosenwald, M.A. Basson, J.D. Licht, J.T.R. Pena, S.H. Rouhanifard, M.U. Muckenthaler, T. Tuschl, G.R. Martin, J. Bauersachs, S. Engelhardt, Nature 456 (2008) 980–984. [2] I.A. Asangani, S.A.K. Rasheed, D.A. Nikolova, J.H. Leupold, N.H. Colburn, S. Post, H. Allgayer, Oncogene 27 (2008) 2128–2136. [3] P.P. Medina, M. Nolde, F.J. Slack, Nature 467 (2010) 86–90. [4] J. Du, S. Yang, D. An, F. Hu, W. Yuan, C. Zhai, T. Zhu, Cell Res. 19 (2009) 487–496. [5] S. Zhu, H. Wu, F. Wu, D. Nie, S. Sheng, Y.Y. Mo, Cell Res. 18 (2008) 350–359. [6] T. Li, D. Li, J. Sha, P. Sun, Y. Huang, Biochem. Biophys. Res. Commun. 383 (2009) 280–285. [7] S. Asaga, C. Kuo, T. Nguyen, M. Terpenning, A.E. Giuliano, D.S.B. Hoon, Clin. Chem. 57 (2011) 84–91. [8] L. Yu, N.W. Todd, L. Xing, Y. Xie, H. Zhang, Z. Liu, H.B. Fang, J. Zhang, R.L. Katz, F. Jiang, Int. J. Cancer 127 (2010) 2870–2878. [9] K. Shi, B. Dou, C. Yang, Y. Chai, R. Yuan, Y. Xiang, Anal. Chem. 87 (2015) 8578–8583. [10] S.J. Zhen, X. Xiao, C.H. Li, C.Z. Huang, Anal. Chem. 89 (2017) 8766–8771. [11] X. Liu, S.Q. Zhang, Z.H. Cheng, X. Wei, T. Yang, Y.L. Yu, M.L. Chen, J.H. Wang, Anal. Chem. 90 (2018) 12116–12122. [12] J. Park, J.S. Yeo, Chem. Commun. 50 (2014) 1366–1368. [13] L. Liu, C. Song, Z. Zhang, J. Yang, L. Zhou, X. Zhang, G. Xie, Biosens. Bioelectron. 70 (2015) 351–357. [14] H.L. Shuai, K.J. Huang, Y.X. Chen, L.X. Fang, M.P. Jia, Biosens. Bioelectron. 89 (2017) 989–997. [15] H. Han, E. Livingston, X. Chen, Anal. Chem. 83 (2011) 8184–8191. [16] Z. Chen, Q. Li, Q. Sun, H. Chen, X. Wang, N. Li, M. Yin, Y. Xie, H. Li, B. Tang, Anal. Chem. 84 (2012) 4687–4694. [17] S. Jin, G.J. Anderson, R.T. Kennedy, Anal. Chem. 85 (2013) 6073–6079. [18] A.J. Gaudry, Y.H. Nai, R.M. Guijt, M.C. Breadmore, Anal. Chem. 86 (2014) 3380–3388. [19] H. Li, Q. Li, X. Wang, K. Xu, Z. Chen, X. Gong, X. Liu, L. Tong, B. Tang, Anal. Chem.
3.6. Detection of miR-21 in human cells The established method was applied to detect miR-21 in human cells. The lysate of human breast cancer cells (MCF-7), human bladder cancer cells (T24) and human bladder epithelial cells (HCV-29) were selected for the samples. The cell samples were treated according to the experimental method, and the cell lysate was diluted with electrophoresis buffer for 25-fold and analyzed under the optimized experimental conditions. Five parallel experiments were carried out and the electrophoretograms are shown in Fig. S4. Since miR-21 is overexpressed in cancer cells, the peaks of HRP-labeled DNA fragments can be easily seen of MCF-7 cells and T24 cells compare with that of HCV29 cells. The analysis results are shown in Table 1, the contents of miR21 are about 139 fM in the lysate of MCF-7 cell and about 58 fM in T24 cells, but miR-21 was not detected in HCV-29 cells due to its low concentration. The detection results are similar with previous reports about the miR-21 content in cancer and normal cells [33,34]. The recovery is between 98.8% and 109.2%, and the relative standard deviation (RSD) of five parallel experiments is less than 5%. These results suggest that the developed assay can be used for the recognition of cancer cells and healthy cells. 5
Talanta xxx (xxxx) xxxx
C. He, et al. 81 (2009) 2193–2198. [20] Z. Chen, Q. Li, X. Wang, Z. Wang, R. Zhang, M. Yin, L. Yin, K. Xu, B. Tang, Anal. Chem. 82 (2010) 2006–2012. [21] M.K. Hulvey, C.N. Frankenfeld, S.M. Lunte, Anal. Chem. 82 (2010) 1608–1611. [22] L. Li, Q. Li, P. Chen, Z. Li, Z. Chen, B. Tang, Anal. Chem. 88 (2016) 930–936. [23] S. Zhao, X. Li, Y.M. Liu, Anal. Chem. 81 (2009) 3873–3878. [24] M.R. Mohamadi, Z. Svobodova, R. Verpillot, H. Esselmann, J. Wiltfang, M. Otto, M. Taverna, Z. Bilkova, J.L. Viovy, Anal. Chem. 82 (2010) 7611–7617. [25] C.M. Snyder, W.R. Alley Jr., M.I. Campos, M. Svoboda, J.A. Goetz, J.A. Vasseur, S.C. Jacobson, M.V. Novotny, Anal. Chem. 88 (2016) 9597–9605. [26] I. Mitra, Z. Zhuang, Y. Zhang, C.Y. Yu, Z.T. Hammoud, H. Tang, Y. Mechref, S.C. Jacobson, Anal. Chem. 84 (2012) 3621–3627. [27] T. Kawai, K. Sueyoshi, F. Kitagawa, K. Otsuka, Anal. Chem. 82 (2010) 6504–6511.
[28] Z.F. Wang, S. Cheng, S.L. Ge, H. Wang, Q.J. Wang, P.G. He, Y.Z. Fang, Anal. Chem. 84 (2012) 1687–1694. [29] L. Li, Q. Wang, J. Feng, L. Tong, B. Tang, Anal. Chem. 86 (2014) 5101–5107. [30] M. Shi, Y. Huang, J. Zhao, S. Li, R. Liu, S. Zhao, Talanta 179 (2018) 466–471. [31] J. Li, J. Zhao, S. Li, L. Zhang, Y. Huang, S. Zhao, Y.M. Liu, Chem. Commun. 52 (2016) 12806–12809. [32] L. Li, P. Li, J. Fang, Q. Li, H. Xiao, H. Zhou, B. Tang, Anal. Chem. 87 (2015) 6057–6063. [33] Y. Liao, R. Huang, Z. Ma, Y. Wu, X. Zhou, D. Xing, Anal. Chem. 86 (2014) 4596–4604. [34] J. Zhang, D. Wu, Q. Chen, M. Chen, Y. Xia, S. Cai, X. Zhang, F. Wu, J. Chen, Analyst 140 (2015) 5082–5089.
6
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Ultrasensitive detection of microRNA-21 based on electrophoresis assisted cascade chemiluminescence signal amplification for the identification of cancer cells Caimei Hea, Shengyu Chena, Jingjin Zhaoa,b,∗, Jianniao Tiana, Shulin Zhaoa a b
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University, Guilin, 541004, China Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection, Ministry of Education, Guangxi Normal University, Guilin, 541004, China
ARTICLE INFO
ABSTRACT
Keywords: miRNA detection Microchip electrophoresis Signal amplification Chemiluminescence
Rapid and accurate detection of microRNA content in cells is of great significance. Here, an ultrasensitive microchip electrophoresis (MCE) method based on cascade chemiluminescence (CL) signal amplification was developed for the detection of microRNA-21 in cells. In this method, horseradish peroxidase labeled DNA was used as a signal probe, which could induce CL signal by the reaction of luminol and H2O2. Combining with two cyclic enzyme digestion reactions by T7 exonuclease, a large number of signal probes were degraded. By using MCE-CL as a separation and detection platform, an amplified CL signal peak was achieved. The developed MCE-CL method can detect miR-21 at a concentration as low as 1.0 × 10−15 M, which was enhanced by six orders of magnitude compared with those of conventional MCE-CL assay. This method has been applied for the detection of microRNA-21 in cell lysate, which show that there were significant differences of miR-21 among different types of cells, and the content in cancer cells was much higher than that in normal cells, which can be used for the identification of cancer cells. Therefore, the proposed method held great application potential in early diagnosis of tumor and biomedical research.
1. Introduction MicroRNA-21 (miR-21) is a small RNA molecule with high expression in tumor. It has been found that human miR-21 is located in the 17q23.2 region of chromosome, and is related to many diseases [1]. The miR-21 is a carcinogenic miRNA, which is the only high expression miRNA found in multiple solid and non-solid tumors [2,3]. As an important miRNA with tumor gene properties, miR-21 can regulate the growth of breast cancer cells [4]. Its gene cluster can promote many kinds of tumors, and it has similar oncogene function in tumorigenesis and development. It also involved in tumor angiogenesis, and closely related to tumor cell proliferation, invasion and drug resistance [5,6]. In recent years, miR-21 may become a new biomarker for early diagnosis and gene therapy with the further study of its function [7,8]. Therefore, accurate detection of miR-21 in cells is of great significance for early diagnosis of related diseases and the discovery of new anticancer drugs. The content of miR-21 in human body is very low, raising the difficulties to detect its concentration with traditional methods. Moreover,
the most urgently need is to detect miRNA accurately in a complex biological matrix. A series of analytical methods have been developed for the determination of miR-21 by using various techniques to improve the sensitivity and selectivity. For example, Shi et al. reported an enzyme-free target recycling signal amplification electrochemical sensor [9]. Zhen et al. proposed an enzyme-free DNA circuit-assisted graphene oxide enhanced fluorescence anisotropy assay [10]. Liu et al. developed an inductively coupled plasma mass spectrometry method for the determination of miR-21 in human plasma [11]. Park et al. proposed a colorimetric sensing method on the basis of the plasmonic coupling effect [12]. Liu et al. developed an electrochemical analysis method based on the layer-by-layer assembly of oxidized single-walled carbon nanotubes and nanodiamonds on the electrode [13], and Shuai et al. proposed a nanocrystalline gold/hollow molybdenum disulfide micro cube biosensor [14]. However, most of these methods are cumbersome, require a large volume of sample or with high background. To this end, the development of simple, rapid, low-consumption and high-sensitivity analytical methods for miR-21 detection is still a subject of high attention.
∗ Corresponding author. State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University, Guilin, 541004, China. E-mail address: [email protected] (J. Zhao).
https://doi.org/10.1016/j.talanta.2019.120505 Received 14 June 2019; Received in revised form 21 October 2019; Accepted 24 October 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Caimei He, et al., Talanta, https://doi.org/10.1016/j.talanta.2019.120505
Talanta xxx (xxxx) xxxx
C. He, et al.
As a new separation and analysis platform, the microchip electrophoresis (MCE) has the advantages of low sample and reagent consumption, fast analytical speed, high throughput and automation [15–18]. In recent years, MCE technique has been applied in biochemical [19–21], single cell [22,23] and clinical analysis [24–26]. However, the sensitivity of conventional MCE analysis is relatively low because of its extremely small sample size, which limits its application in the detection of trace substances in organisms. To overcome this drawback, a variety of methods have been proposed to improve the sensitivity of MCE technology. For example, kawai et al. proposed a large-volume sample stacking method, and the sensitivity was increased 2200–2900 times [27]. Wang et al. proposed a multiple-concentration approach combining chitosan sweeping, field-amplified sample stacking, and reversed-field stacking, with the sensitivity increasing 6000 times [28]. Tang's team put forward the strategy of nucleic acid adaptation and enzyme-assisted signal amplification, which made the detection limit of microchip platform reach 1.9 × 10−11 M [29]. Although the sensitivity has been improved a lot by these methods, it is still a challenge to realize the accurate determination of miRNA on the MCE platform due to its low content in organisms. Chemiluminescence (CL) is a powerful and widely used analytical technique with merits of low background, high sensitivity and wide linear range. Coupling with CL detection, MCE-CL method has attracted great attention for its good performance in analysis [30]. Some of them have been successfully used for detection of DNA fragments, enzymes and proteins, which indicates that CL is a powerful and promising technique for MCE assay. The present work integrates the advantages of MCE technology and CL reaction of between luminol and hydrogen peroxide (H2O2), combining with two cyclic enzyme digestion reaction of DNA probes by T7 exonuclease (T7exo). An electrophoresis assisted cascade CL signal amplification method was developed for ultrasensitive detection of miRNA-21. The limit of detection of this MCE-CL platform reached 1.0 × 10−15 M. This strategy has been applied for the detection of miRNA-21 in cell lysate, and realize the recognition of miR-21 in both cancer cells and normal cells.
(Shanghai Precision Scientific Instrument Co. Ltd), SH-III water circulating vacuum pump (Zhengzhou Great Wall Scientific Industrial Co. Ltd), and FS-150N ultrasonic instrument (Shanghai shengxi ultrasonic instrument Co., Ltd). 2.2. Cascade amplification reaction The single-stranded DNA probe H1 and H2 were diluted to 2 μM with Tris-HCl buffer respectively, and then heated at 95 °C for 5 min, followed by cooling to room temperature naturally. After that, 4 μL of H1 and H2, 2 μL of different concentration of miRNA, 1.5 μL of 10 × NEBbuffer 4 and 6.5 μL of Tris-HCl buffer were added to a 100 μL centrifuge tube, respectively. Then 4 μL of 1.6 × 10−7 M HRP-DNA probe and 3 μL of 10 U/μL enzyme T7exo were added in the above solution. After mixed and incubated at 37 °C for 120 min in a water bath, 29 μL of Tris-HCl buffer was added to a volume of 50 μL before MCE-CL analysis. 2.3. Microchip electrophoresis analysis Before new chip was used, the channels were flushed with 10 mM SDS and methanol solution for 5 min each, then rinsed with 1 M hydrochloric acid for 10 min, 0.1 M sodium hydroxide for 30 min. Between two sample runs, the channel was sequentially activated with 0.1 M NaOH solution for 10 min, rinsed with water for 5 min. Subsequently, each reservoir was filled with electrophoresis buffer, and filled chip channels using a vacuum pump. Then, the sample solution and CL buffer solution were used to replace the solution in the S and the R reservoirs, respectively. The sample solution was injected into the separation channel in pinched mode by applying 500 V, 250 V and 400 V voltages to S, B and BW reservoirs. SW reservoir grounding and R reservoir was suspended. After 20 s, the voltage of each reservoir was switched, 2400 V was applied to the B reservoir as the separation voltage, 1500 V voltage was applied to the S and SW as the pull back voltage, while the BW reservoir was grounded, and 500 V voltage was applied in the R reservoir. Under the action of electroosmotic flow, the analytes migrate to the Y-type intersection at different rates in the separation channel, and mixed with the oxidizer to generate CL signal, which were collected by the photomultiplier tube (PMT).
2. Experimental 2.1. Reagents and apparatus
2.4. Preparation of cells lysate
The nucleic acid molecules used in this study were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China), which are shown in Table S1. Luminol was purchased from Fluka Co., Ltd. (Switzerland), and the P-piphenol (PIP) was obtained from Shanghai Bangcheng Chemical Co., Ltd. (China). H2O2, sodium dodecyl sulfate (SDS) and borax were purchased from Shanghai Chemical Reagent Co., Ltd (Shanghai, China). Trimethylamino methane (Tris) was achieved from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). T7 exonuclease (T7exo) and 10 × NEB buffer 4 were purchased from New England Biolabs Inc. (Beverly, MA, USA). Other chemicals used in this work were of analytical grade. Ultrapure water (18.2 MΩ) was used throughout the experiments. The solutions used in this work include: DNA storage solution (2.0 × 10−5 M in 1.0 × 10−2 M Tirs-EDTA buffer, pH = 8); TirsHCl buffer (1.0 × 10−2 M Tirs-HCl, 20 mM KCl, 0.2 M NaCl, pH = 7.4); electrophoresis buffer (25 mM borate, 15 mM SDS, 1.4 mM luminol, pH = 9.4) and CL reaction buffer (35 mM NaHCO3, 130 mM H2O2, pH = 10.5). MCE analysis was carried out on a self-assembled MCE-CL detection system by our laboratory [31]. The versatile programmable eight-pathelectrode power supply was developed and provided by Shandong Normal University [32]. Chromatography data system was purchased by Zhejiang University Zhida Information liability Co., Ltd. The chip used in this work is double T-type glass microchip with broadened Ytype CL detection cell, which was fabricated by Dalian Tuowei Technology Co., Ltd. The dimensions of the microchip was shown in Fig. S1. Other instruments used in this study include PHSJ-4A pH meter
The cells used in the experiment are human breast cancer cells (MCF-7), bladder cancer cells (T24) and normal human bladder cells (SV-HUC-1). The cell lines were cultured in a DMEM culture medium containing 10% fetal bovine serum, 10 U mL−1 penicillin, 10 g mL−1 streptomycin, and incubated at 37 °C for 24 h in a 5% CO2 atmosphere. After digesting with trypsin, the culture medium was removed by centrifugation, and the cells were dispersed in the PBS buffer. This procedure was repeated three times. Then, the cells were resuspended in 10 mM Tris-HCl buffer (pH = 7.4) with a cell density of about 1.0 × 104 cells mL−1. A 3 mL volume of above cell suspension was added into the centrifuge tube, and centrifuging for 5 min at a speed of 1000 r/min. Then, the PBS solution was abandoned, 200 μL Tris-HCl buffer solutions were used to suspend the cells, and transfer into a 1.5 mL centrifuge tube. The centrifugal tube was put in ice water bath on the ultrasonic instrument, with 80 W ultrasonic power ultrasonic broken for 20 min. After ultrasonic fragmentation, ultrafiltration membranes with a molecular weight cutoff of 10 kDa were used to remove large molecular weight proteins and extract the filtrate was retained for analysis. 2.5. Gel electrophoresis Agarose gel electrophoresis tests were carried out to verify the hybridization of miR-21, H1 and H2, and cleavage was induced by T7exo. 2
Talanta xxx (xxxx) xxxx
C. He, et al.
Fig. 1. Schematic diagram for MCE method based on separation assisted cascade CL signal amplification. Fig. 2. (A) The electrophoretograms for feasibility verification. (a) 0.16 μM HRPDNA, (b) 0.16 μM HRP-DNA+100 fM miR21, (c) 0.16 μM HRP-DNA+1 pM miR-21, (d) 0.16 μM HRP-DNA+10 pM miR-21. Peak identification: 1. HRP-DNA, 2. HRPlabeled monucleotide fragment. (B) Agarose gel electrophoresis characterization. Lane 1: miR-21.2: HRP-DNA. 3: H1/H2. 4: HRPDNA + H1/H2. 5: H1/H2+miR-21.6: H1/ H2+HRP-DNA + T7exo. 7: H1/H2+miR21+HRP-DNA + T7exo. Concentrations of HRP-DNA, H1/H2, miR-21 and T7exo are 3 μM, 3 μM, 2 μM and 40 U, respectively.
First, 4 g agarose was mixed with 100 mL of 0.5 × TBE and the mixture was heated by microwave for 3 min to produce agarose gel. Before electrophoresis, SYBR Green I, SYBR Green II was added to singlestranded DNA, double-stranded DNA samples and incubated for 10 min. After 4 μL of loding buffer was added, 60 min electrophoresis was performed at 100 V constant voltage. The electrophoresis results were observed using the Omega-16ic imaging system and photographed for analysis.
with the signal probe HRP-DNA to form a H2/HRP-DNA duplex, which could also be degraded by T7exo to release H2. The released H2 hybridized with another HRP-DNA probe to initiate the next cycle. As a result, amounts of HRP-labeled single-stranded DNA fragments were produced during the degradation process by T7exo. When CL signal was made through the HRP and luminol-H2O2 reaction, a cascade signal amplification could be achieved. The HRP-labeled DNA fragments and undigested HRP-DNA probes were separated and two peaks were present in the electropherogram after the MCE separation. The peak height of the HRP-labeled single-stranded DNA fragments was used for the quantification of miR-21.
3. Results and discussion 3.1. Method design and principle
3.2. Feasibility study of the method
The working principle of the method is shown in Fig. 1. First, DNA probe H1 hybridized with H2 to form a partially complementary duplex H1/H2. The prominent 5’ ends of DNA probes could prevent degradation by T7exo in the absence of target miR-21. T7exo is a doublestranded DNA specific exonuclease which can catalyze the removal of nucleotides from linear or nicked double-stranded DNA in the 5′ to 3′ direction. So, when miR-21 hybridized with H1 probe, T7exo recognized and degraded the duplex H1/H2 along the 5′→3′ direction of DNA sequence, releasing the target (miR-21) and H2 probe. Then, the released miR-21 continued to hybridize with remaining H1/H2 and triggered N cycles digestion of H1 probe by T7exo, resulting in the release of large numbers of H2 probes. The displaced free H2 hybridized
In order to verify the feasibility of the method, the effects of different miR-21 concentrations on the peak intensity of the HRP-labeled DNA fragments was investigated, and the experimental results are shown in Fig. 2A. In the absence of miR-21, the resulting electrophoresis curve a showed only one electrophoresis peak in the MCE-CL test. When a trace of miR-21 was added, the enzyme-assisted cascade amplification reaction was initiated and HRP-labeled DNA fragments were produced. The positions of the two peaks are different due to the difference of charge-to-mass ratio between the HRP-labeled DNA fragment and the HRP-DNA probe. The charge-to-mass ratio of the former is higher than that of the latter, so that the peak of HRP-labeled DNA 3
Talanta xxx (xxxx) xxxx
C. He, et al.
Fig. 3. (A) Electrophoretograms for detection of miR-21 in the concentration range of 5.0 fM~10 nM. (B) Dynamic curve of miR-21 detection (inset is linear curve of miR-21 detection). CL reaction condition: 120 mM H2O2; 1.2 mM luminol; 1.0 mM PIP; CL buffer pH = 10.5; 0.16 μM HRP-DNA; 30 U T7exo. MCE separation condition: 20 mM borax; 15 mM SDS; electrophoresis buffer pH = 10; voltage 2400 V. Peak identification: 1. HRP-DNA, 2. HRP-labeled nucleotide fragments.
fragment is firstly presented in curve b, c and d. With the increase of the amount of miR-21, the amount of the HRP-labeled DNA fragment was also increased, and the intensity of peak 2 gradually enhanced, while the intensity of peak 1 for the HRP-DNA probe decreased. The experimental results indicated that the proposed method is feasible for miR21 detection. Agarose gel electrophoresis tests were carried out on different systems to further verify the feasibility. As shown in Fig. 2B, two bands were observed in lane 4 which contained the mixture of H1/H2 and HRP-DNA, indicating that HRP-DNA could not hybridize with H2 in the absent of taget. When miR-21 was added with H1/H2, only one band in a backward position could be seen in lane 5, and a fully complementary double-stranded DNA was formed. Compare with lane 4, T7exo was added in lane 6, the two bands in the same position were still present because T7exo can not degrade the H1/H2 duplex without target. In the present of miR-21, the band of H1/H2 duplex was disappeared in lane 7, which means that H1 probe was degraded by T7exo. The above results prove the feasibility of the method again.
buffer, the concentration of SDS and borax in the electrophoresis buffer. The electrophoresis separation voltage in the range of 2300–2500 V was examined in Fig. S3A. When the voltage increases from 2300 V up to 2400 V, the resolution (Rs) of the sample increases gradually, and the Rs decreases when the voltage continues to increase, which may be due to the excessive Joule heat generated when the voltage is too large, and the bubbles are produced during the electrophoresis process. Therefore, the optimal separation voltage is selected as 2400 V. The effect of pH value of electrophoresis buffer solution in the range of 9.0–11.0 on the Rs are shown in Fig. S3B, and pH = 10 is chosen as the optimal pH value of electrophoresis buffer solution. SDS is an anionic surfactant which is commonly used in the separation of proteins and lipids. It can improve the electrophoretic separation. In our work, when the concentration of SDS was 15 mM, the Rs reaches the maximum value (Fig. S3C). The effect of borax concentration in the concentration range of 10–30 mM on the samples separation are shown in Fig. S3D The Rs reached the maximum value at 20 mM and then decrease gradually as the concentration of borax continues to increase, which is due to the excessive Joule heat caused by the excessive ion concentration of electrophoretic buffer solution.
3.3. Optimization of experimental conditions Several experimental conditions were investigated to achieve a better perfomance, including the concentrations of luminol, H2O2 and PIP, the pH value, T7exo dosage and reaction time. As shown in Figs. S2A and S2B, CL intensity reached the maximum when the concentrations of luminol and H2O2 are 1.2 mM and 120 mM, respectively. The sensitivity of the CL system can be improved by using CL sensitizers such as phenols. PIP was used as the sensitizer in our work, and the effect of PIP in the concentration range of 0.6–1.5 mM on the CL intensity was investigated. When the PIP concentration was 1.0 mM, the maximum CL intensity was obtained (Fig. S2C). Fig. S2D show the effect of the pH value of post-column buffer solution on the CL intensity, and pH = 10.5 was selected for the best pH value of the buffer solution. The effect of T7exo concentration in the range of 10–40 U on the CL intensity is shown in Fig. S2E. When the concentration of T7exo increased from 10 U to 30 U, the CL intensity of HRP-labeled monucleotide fragment increased gradually and then remained basically unchanged. The optimal concentration of T7exo in this study was 30 U. The effect of reaction time is presented in Fig. S2F, after the reaction time is longer than 140 min, the CL intensity almost keep constant. Therefore, the optimized reaction time is 140 min. The MCE separation conditions were also optimized, such us the electrophoresis separation voltage, the pH value of the electrophoresis
3.4. The assay performance for miR-21 detection Under the optimized experimental conditions, the analytical performances of this assay were investigated, and the experimental results are shown in Fig. 3. The peak height of the HRP-labeled DNA fragments raises gradually with the increase concentration of miR-21 (Fig. 3A, peak 2). As seen in Fig. 3B, it has a good linear relationship between peak signal and the logarithmic value of miR-21 concentration in the range from 5.0 fM to 10 nM. The linear equation is Y = 1.943X +11.664. Where Y represents the peak 2 height (mV), X represents the logarithmic value of miR-21 concentration, and the correlation coefficient R2 is 0.9967. The detection limit was calculated to be 1.0 fM with S/N = 3. 3.5. Study on the method specificity The miRNA is a kind of short-chain RNA with high sequence homology. In order to determine the target miRNA accurately, the specificity of the method must be investigated. Therefore, the effects of single base mismatch SM-21 and other miRNAs such as miR-15, miR-16 were investigated, the results are shown in Fig. 4. Compared with miR21, the CL intensity of other miRNAs are similar to that of the blank 4
Talanta xxx (xxxx) xxxx
C. He, et al.
4. Conclusions In summary, an ultrasensitive MCE method based on cascade CL signal amplification was developed for the detection of microRNA-21. As far as we know, this is the first cascade CL signal amplification method based on MCE-CL platform for the detection of miR-21. The advantages of this assay include simple operation, high degree of automation, short analysis time, low consumption of reagents and samples, very high sensitivity and good specificity. The detection limit for miR-21 was enhanced by six orders of magnitude compared to those of conventional MCE-CL method. This assay has been applied for the detection of miR-21 in cell lysate, and results show that there were significant differences of miR-21 among different types of cells. The content in cancer cells was much higher than that in normal cells, which can be used for the identification of cancer cells. Therefore, it has a wide application prospect in the early diagnosis of tumors and biomedical research. Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled.
Fig. 4. Study on the specificity of the method. The concentration of miR-21 is 6.0 pM. The concentrations of SM-21, miR-15, miR-16 and miR-141 are 600 pM, respectively. The experimental conditions are the same as in Fig. 3.
Table 1 The results for the detection of miR-21 in human cells. Samples
Founda (pM)
Added (pM)
Total found (pM)
RSD (%,N = 5)
Recovery (%)
MCF-7
0.139
T24
0.058
HCV-29
ND
0.250 0.500 0.750 0.250 0.500 0.750 0.250 0.500 0.750
0.412 0.642 0.884 0.315 0.552 0.822 0.260 0.504 0.764
2.7 1.6 1.1 2.8 1.7 1.6 2.6 2.0 1.6
109.2 100.6 99.3 102.8 98.8 101.8 104.0 100.8 101.8
a
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21765003), Natural Science Foundation of Guangxi Province (No. 2017GXNSFFA198014) and BAGUI Scholar Program. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2019.120505.
ND: No miR-21 was detected.
References
under the same experimental conditions, and the CL intensity of mismatch SM-21 is a quarter of that with target miR-21. The aboved results indicated that proposed method has a high specificity.
[1] T. Thum, C. Gross, J. Fiedler, T. Fischer, S. Kissler, M. Bussen, P. Galuppo, S. Just, W. Rottbauer, S. Frantz, M. Castoldi, J. Soutschek, V. Koteliansky, A. Rosenwald, M.A. Basson, J.D. Licht, J.T.R. Pena, S.H. Rouhanifard, M.U. Muckenthaler, T. Tuschl, G.R. Martin, J. Bauersachs, S. Engelhardt, Nature 456 (2008) 980–984. [2] I.A. Asangani, S.A.K. Rasheed, D.A. Nikolova, J.H. Leupold, N.H. Colburn, S. Post, H. Allgayer, Oncogene 27 (2008) 2128–2136. [3] P.P. Medina, M. Nolde, F.J. Slack, Nature 467 (2010) 86–90. [4] J. Du, S. Yang, D. An, F. Hu, W. Yuan, C. Zhai, T. Zhu, Cell Res. 19 (2009) 487–496. [5] S. Zhu, H. Wu, F. Wu, D. Nie, S. Sheng, Y.Y. Mo, Cell Res. 18 (2008) 350–359. [6] T. Li, D. Li, J. Sha, P. Sun, Y. Huang, Biochem. Biophys. Res. Commun. 383 (2009) 280–285. [7] S. Asaga, C. Kuo, T. Nguyen, M. Terpenning, A.E. Giuliano, D.S.B. Hoon, Clin. Chem. 57 (2011) 84–91. [8] L. Yu, N.W. Todd, L. Xing, Y. Xie, H. Zhang, Z. Liu, H.B. Fang, J. Zhang, R.L. Katz, F. Jiang, Int. J. Cancer 127 (2010) 2870–2878. [9] K. Shi, B. Dou, C. Yang, Y. Chai, R. Yuan, Y. Xiang, Anal. Chem. 87 (2015) 8578–8583. [10] S.J. Zhen, X. Xiao, C.H. Li, C.Z. Huang, Anal. Chem. 89 (2017) 8766–8771. [11] X. Liu, S.Q. Zhang, Z.H. Cheng, X. Wei, T. Yang, Y.L. Yu, M.L. Chen, J.H. Wang, Anal. Chem. 90 (2018) 12116–12122. [12] J. Park, J.S. Yeo, Chem. Commun. 50 (2014) 1366–1368. [13] L. Liu, C. Song, Z. Zhang, J. Yang, L. Zhou, X. Zhang, G. Xie, Biosens. Bioelectron. 70 (2015) 351–357. [14] H.L. Shuai, K.J. Huang, Y.X. Chen, L.X. Fang, M.P. Jia, Biosens. Bioelectron. 89 (2017) 989–997. [15] H. Han, E. Livingston, X. Chen, Anal. Chem. 83 (2011) 8184–8191. [16] Z. Chen, Q. Li, Q. Sun, H. Chen, X. Wang, N. Li, M. Yin, Y. Xie, H. Li, B. Tang, Anal. Chem. 84 (2012) 4687–4694. [17] S. Jin, G.J. Anderson, R.T. Kennedy, Anal. Chem. 85 (2013) 6073–6079. [18] A.J. Gaudry, Y.H. Nai, R.M. Guijt, M.C. Breadmore, Anal. Chem. 86 (2014) 3380–3388. [19] H. Li, Q. Li, X. Wang, K. Xu, Z. Chen, X. Gong, X. Liu, L. Tong, B. Tang, Anal. Chem.
3.6. Detection of miR-21 in human cells The established method was applied to detect miR-21 in human cells. The lysate of human breast cancer cells (MCF-7), human bladder cancer cells (T24) and human bladder epithelial cells (HCV-29) were selected for the samples. The cell samples were treated according to the experimental method, and the cell lysate was diluted with electrophoresis buffer for 25-fold and analyzed under the optimized experimental conditions. Five parallel experiments were carried out and the electrophoretograms are shown in Fig. S4. Since miR-21 is overexpressed in cancer cells, the peaks of HRP-labeled DNA fragments can be easily seen of MCF-7 cells and T24 cells compare with that of HCV29 cells. The analysis results are shown in Table 1, the contents of miR21 are about 139 fM in the lysate of MCF-7 cell and about 58 fM in T24 cells, but miR-21 was not detected in HCV-29 cells due to its low concentration. The detection results are similar with previous reports about the miR-21 content in cancer and normal cells [33,34]. The recovery is between 98.8% and 109.2%, and the relative standard deviation (RSD) of five parallel experiments is less than 5%. These results suggest that the developed assay can be used for the recognition of cancer cells and healthy cells. 5
Talanta xxx (xxxx) xxxx
C. He, et al. 81 (2009) 2193–2198. [20] Z. Chen, Q. Li, X. Wang, Z. Wang, R. Zhang, M. Yin, L. Yin, K. Xu, B. Tang, Anal. Chem. 82 (2010) 2006–2012. [21] M.K. Hulvey, C.N. Frankenfeld, S.M. Lunte, Anal. Chem. 82 (2010) 1608–1611. [22] L. Li, Q. Li, P. Chen, Z. Li, Z. Chen, B. Tang, Anal. Chem. 88 (2016) 930–936. [23] S. Zhao, X. Li, Y.M. Liu, Anal. Chem. 81 (2009) 3873–3878. [24] M.R. Mohamadi, Z. Svobodova, R. Verpillot, H. Esselmann, J. Wiltfang, M. Otto, M. Taverna, Z. Bilkova, J.L. Viovy, Anal. Chem. 82 (2010) 7611–7617. [25] C.M. Snyder, W.R. Alley Jr., M.I. Campos, M. Svoboda, J.A. Goetz, J.A. Vasseur, S.C. Jacobson, M.V. Novotny, Anal. Chem. 88 (2016) 9597–9605. [26] I. Mitra, Z. Zhuang, Y. Zhang, C.Y. Yu, Z.T. Hammoud, H. Tang, Y. Mechref, S.C. Jacobson, Anal. Chem. 84 (2012) 3621–3627. [27] T. Kawai, K. Sueyoshi, F. Kitagawa, K. Otsuka, Anal. Chem. 82 (2010) 6504–6511.
[28] Z.F. Wang, S. Cheng, S.L. Ge, H. Wang, Q.J. Wang, P.G. He, Y.Z. Fang, Anal. Chem. 84 (2012) 1687–1694. [29] L. Li, Q. Wang, J. Feng, L. Tong, B. Tang, Anal. Chem. 86 (2014) 5101–5107. [30] M. Shi, Y. Huang, J. Zhao, S. Li, R. Liu, S. Zhao, Talanta 179 (2018) 466–471. [31] J. Li, J. Zhao, S. Li, L. Zhang, Y. Huang, S. Zhao, Y.M. Liu, Chem. Commun. 52 (2016) 12806–12809. [32] L. Li, P. Li, J. Fang, Q. Li, H. Xiao, H. Zhou, B. Tang, Anal. Chem. 87 (2015) 6057–6063. [33] Y. Liao, R. Huang, Z. Ma, Y. Wu, X. Zhou, D. Xing, Anal. Chem. 86 (2014) 4596–4604. [34] J. Zhang, D. Wu, Q. Chen, M. Chen, Y. Xia, S. Cai, X. Zhang, F. Wu, J. Chen, Analyst 140 (2015) 5082–5089.
6