Quantitative Detection of PremicroRNA-21 Based on Chimeric Molecular Beacon

CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 46, Issue 7, July 2018 Online English edition of the Chinese language journal

Cite this article as: Chinese J. Anal. Chem., 2018, 46(7): e1847–e1853

RESEARCH PAPER

Quantitative Detection of PremicroRNA-21 Based on Chimeric Molecular Beacon XIE Hai-Long1, WU Tian-Tian1, XIE Zhen-Hua2, FAN Jia-Long2, TONG Chun-Yi2,* 1 2

Institute of Cancer Research, School of Medicine, University of South China, Hengyang 421001, China; College of Biology, Hunan University, Changsha 410082, China

Abstract: As an important tumor marker, the expression level of premicroRNA-21 (pre-miR-21) can provide important information for early diagnosis, drug treatment and prognosis of tumor. Thus, developing pre-miR-21 detection is of great importance for diagnosis of disease. Herein, a direct, simple and rapid method for quantitative detection of pre-miR-21 was developed. This detecting system contained the tool of molecular beacon which could hybridize specifically with pre-miR-21 and form duplex to induce fluorescence signal change. When loop bases hybridized with the pre-miR-21 in the solution to form a double-stranded duplex, the fluorophore and the quencher was separated and caused fluorescence recovery. We developed the new method for pre-miR-21 assay with detection limit of 0.5 nM. Under the optimal conditions, the method was used to detect the level of pre-miR-21 in different tumor cells and tissues. The results showed that pre-miR-21 level was tightly related with tumor cells origins and malignancy degree. In 16 cases of gastric cancer tissues and adjacent tissues, the level of pre-miR-21 in 12 cases of cancer tissues was higher than that of adjacent tissues; 3 cases had lower expression level than that of adjacent tissues, and 1 case had no significant difference. Furthermore, Quantitative real-time PCR (qRT-PCR) method was used in to confirm the reliability of the detection results. The consistent results of the two different methods clearly showed that the molecular beacon assay with advantages of simplicity and rapidity for pre-miR-21 detection was hopeful for the wide usability in the function research and clinical diagnosis of pre-microRNA. Key Words: Chimeric molecular beacon; Pre-microRNA-21; Gastric carcinoma; Quantitative real-time PCR

1

Introduction

Hairpin-structured pre-miRNAs are processed into 60–80 nucleotide (nt) precursor molecules (pre-miRNAs) by the p68Drosha microprocessor complex[1]. Pre-miRNAs are then exported from the nucleus to the cytoplasm by exportin-5[2]. In the cytoplasm, the pre-miRNAs associate with Dicer, which cleaves the pre-miRNA into a miRNA (mature miRNA) approximately 18–24 nt in size[3,4]. The miRNA duplex is then loaded onto Argonaut proteins and presented to the RNAinduced silencing complex (RISC) for the recognition of target mRNAs[5]. Pre-miR-21 as a precursor for overexpression of miRNA-21 in tumors such as gastric cancer, gliomas and so on, can participate in the development of these tumors by

inhibiting the expression of target genes PTEN, PDCD4, RECK, etc[6,7]. Therefore, it is important to develop a sensing technique for quantitative analysis of pre-miR-21 level to study of early diagnosis and pathogenesis of related diseases. Among currently available common pre-miRNAs sensing techniques, such as Northern blot, real-time quantitative PCR[8] and microarrays, are high specific, accurate and sensitive. However, the high demand for samples, complex primer design, time-consuming and other shortcomings still existed in some extent[9]. Therefore, the development of a fast, simple pre-miRNAs detection method still remains an important challenge. Molecular beacons (MB) has been used for in vitro detection of nucleic acid due to the significant advantages of simplicity, rapidity and sensitivity[10–12]. These studies failed to

________________________ Received 18 November 2017; accepted 20 May 2018 *Corresponding author. E-mail: [email protected] This work was supported by the National Natural Science Foundation of China (Nos. 81374062, 81673579, 31672457), the Natural Science Foundation of Hunan Province, China (No. 2018JJ2338), and the Fundamental Research Funds for the Central Universities of China (No. 2015JCA03). Copyright © 2018, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(18)61099-0

XIE Hai-Long et al. / Chinese Journal of Analytical Chemistry, 2018, 46(7): e1847–e1853

fully consider the characteristics of MB that easily formed complex secondary structures and demand higher temperatures for hybridization reactions[13], but direct detection of target molecules using conventional DNA based MB. In early experiments, we found that the stability and background signal of conventional DNA molecular beacons were easily disturbed by external factors such as magnesium ion and reaction temperature[14], thus affecting the reliability of the test results. However, the structure of CMB composed by DNA loop and RNA stem can significantly improve the thermal stability and reduce the background signal as well. In this study, we designed and synthesized CMB according to the sequences of pre-miR-21. After optimizing hybridization conditions of CMB and pre-miR-21, the linear detection range and sensitivity of target molecules were determined using synthetic pre-miR-21 as the target molecule. Based on these considerations, the new method was successfully used for pre-miR-21 assay in biosamples, which indicated the strong anti-interference capability under complicated environment. Furthermore, the detection systems achieved quantitative detection of pre-miR-21 levels in complex biological samples using the established standard curve was realized.

2

Experimental

2.1

Chemicals and materials

All CMBs and oligonucleotide strands (Table 1) were synthesized from Takara Biotechnology Co. Ltd (Dalian, China). The 10 × reactive buffer used in this experiment consisted of 200 mM Tris-HCl (pH 7.8), 400 mM KCl, 100 mM MgCl2, 10 mM DTT, Milli-Q purified water was used to prepare all solutions. All other chemicals were analytical grade without further purification. 2.2

Fluorescence assay

In the hybridization assay, the molecular beacon (MB) and chimeric molecular beacon (CMB) with final concentration of 100 nM were hybridized with target pre-miR-21 in the NE buffer (20 mM Tris-HCl (pH 7.8), 40 mM KCl, 10 mM MgCl2, 1 mM DTT) for 5 min at 95 °C and then 30 min at 45 °C. The fluorescence intensities of solutions were measured using the Hitachi FL-4500 fluorescence spectrophotometer with excitation at 521 nm and emission range from 550 nm to 650

nm. Spectrometer slides were set of 10 nm band-pass. The fitting of the experimental data was accomplished using the software Sigmaplot 12.0. 2.3 Investigation of thermal stability of molecular beacon Comparing the thermal stability between MB and CMB, 1 μL of molecular beacon stock solution (10 μM) with 99 μL NE buffer was mixed respectively. The mixed solution was incubated for 40 min at temperatures of 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 and 80 °C, individually. The fluorescence intensities of the incubated solutions were measured at 578 nm with excitation at 521 nm. 2.4

Feasibility and specificity analysis of CMB detection system

Three samples of CMB/pre-miR-21, CMB/miRNA-21 and CMB were prepared with NE buffer, which the total volume of 100 μL and the final concentrations of CMB, pre-miR-21 and miRNA-21 were 100 nM. The mixed solution of three samples was incubated for 5 min at 95°C, then 30 min at 45°C, respectively. The fluorescence intensities of the three samples were measured at 578 nm with excitation at 521 nm. Finally, the samples were analyzed using PAGE electrophoresis, silver staining and the images were acquired. 2.5

Optimization of influence factors of Mg2+, pH and reaction time

In a final volume of 100 μL solution, 100 nM CMB was separately suspended in the NE buffer and the stability of CMB was investigated by the change of Mg2+ (0, 10, 20, 30, 40 and 50 mM) and pH (5.5–9.0). Then, CMB and pre-mRNA-21 with same concentration of 100 nM were mixed in the NE buffer with final volumes of 100 μL. The recovery of CMB signal was explored by the change of Mg2+, pH and hybridization reaction time. The fluorescence intensity of different reaction conditions was measured as described above. 2.6

Quantitative detection of pre-mRNA-21

In a final volume of 100 μL solution, 100 nM CMB and different concentration pre-mRNA-21 of 0.5, 1, 2, 5, 10, 20,

Table 1 Oligonucleotide strands with modifications Name MB CMB miR-21 pre-miR-21 RNU6B-Primer

Sequences 5’-TMR-CGUUCGACCAUGAGAUUCAACAGTUCGAACG-DABCYL-3’ 5’-TMR-r(CGUUCGA)CCAUGAGAUUCAACAGTr(UCGAACG)-DABCYL-3’ 5’-CAACAUCAGUCUGAUAAGCU-3’ 5’-TGTCGGGTAGCTTATCAGACTGATGTTGACTGTTGAATCTCATGGC AACACCAGTCGATGGGCTGTCTGACA-3’ 5’- CTCGCTTCGGCAGCACA -3’

XIE Hai-Long et al. / Chinese Journal of Analytical Chemistry, 2018, 46(7): e1847–e1853

50 and 100 nM mixed in NE buffer. These mixed solutions were incubated for 5 min at 95 °C, then 30 min at 45 °C, respectively. The fluorescence intensity was measured at 578 nm with excitation at 521 nm. Finally, the standard curve was plotted with the concentration of pre-mRNA-21 (nM) as the X axis and the fluorescence signal at 578 nm as the Y axis. 2.7

Cell culture, total RNA extraction and qRT-PCR

MCF-7 cells were cultured in DMEM medium (Invitrogen) supplemented with 10% fetal calf serum, and penicillinstreptomycin at 37 °C at 5% CO2. Total cellular RNA was isolated from culture cells using TRIzol reagent (Invitrogen) and the concentrations were measured by ultraviolet absorbance at 260 nm and 280 nm and subsequently stored in aliquots at –80 °C. Real-time PCR was performed in the Light Cycler Instrument (Roche Applied Science) in total volume of 200 μL per PCR tube. For each reaction, 1 μL of cDNA was placed in a 20 μL Platinum SYBRgreen qPCR Supermix (Takara) containing 0.1 μL of a temperature-released Taq DNA polymerase (5 U mL–1; Platinum DNA Polymerase; Takara), 0.5 μL of the primers, and 14.5 μL DEPC-treated water. The cycling protocol was identical for pre-mRNA-21 and consisted of an initial 5 min denaturation step at 95 °C for activation of the DNA polymerase, followed by 45 cycles of denaturation at 95 °C for 15 s, annealing at 63 °C for 15 s, and extension at 72 °C for 20 s. 2.8

Polyacrylamide gel electrophoresis and silver staining

Polyacrylamide gel (15%) was prepared by adding 3 mL of ddH2O, 5 mL of 30% acrylamide, 2 mL × 5 Tris-Boric acid (TBE), 70 μL Ammonium perroxodisulfate (APS, 10%) and 4 μL of N,N,N',N'-Tetramethylethylenediamine (TEMED) into centrifuge tube, the mixture was quickly poured into glue. Electrophoresis was carried out at 25 °C for 1 h at a constant volt of 100 V. Finally, the separated products in the gel were visualized by silver staining. 2.9

In this study, we designed and synthesized two hairpin molecular beacons for the detection of pre-miR-21. The loop formed by DNA bases was complementary to the target molecule, and the stem composed of 7 base pairs of DNA (MB) or RNA (CMB), of which 5’end modified with TAMRA and 3’ end was labeled fluorescent quenching group DABCYL. Hairpin molecular beacon acts like a switch that is normally “closed”, the stem structure holds the fluorophore and the quencher in close proximity. Hence, fluorescence quenching occurs as the result of resonance energy transfer[15]. The principle of the MB-based fluorescence assay for pre-miRNA is illustrated in Scheme 1. When loop bases hybridized with the target pre-miRNA in the solution to form a double-stranded duplex, the fluorophore and the quencher are separated and cause fluorescence recovery. The target molecule concentration is determined by intensity of the fluorescence signal. 3.2

Thermal stability of molecular beacons

The thermal stability of MB is reflected by the melting temperature (Tm), which is defined as the temperature that half of hairpin structure is dissociated to single-stranded DNA (ssDNA)[16,17]. Melting temperature measurements are conduct to study the thermal stability of both MB and CMB. Interestingly, the CMB did not open even at 70 °C, as indicated by the constant low fluorescence; while the MB lost its hairpin conformation at temperature above 70 °C (Fig.1). This observation would be attributed to the tight binding of the complementary bases of RNA in the stem. The above results showed that the CMB had better thermal stability than MB, which could not only detect target molecules at higher temperature, but also reduce the background signal. Therefore, CMB further improved the detection sensitivity.

Scheme 1 Schematic presentation of MB-based fluorescence assay for pre-miR-21

Gastric cancer, glioma tissue samples collected and statistics

Gastric cancer, glioma tissue samples were collected in Chenzhou First People's Hospital of Hunan Province and Xiangya Hospital of Central South University, while the samples were treated using liquid nitrogen and preserved in –80 °C refrigerator. Detailed clinical data were shown in supporting information.

3 3.1

Results and discussion Principle of chimeric molecular beacons based assay

Fig.1 Thermo-stability comparison of MB and CMB

XIE Hai-Long et al. / Chinese Journal of Analytical Chemistry, 2018, 46(7): e1847–e1853

3.3

Feasibility of CMB detection system

To examine the ability of CMB to distinguish between different RNA targets, we compared the fluorescence restoration caused by pre-miR-21 and miRNA-21 under the same condition. As demonstrated in Fig.2A, a significant enhancement of fluorescence signal was observed after the addition of pre-miR-21 (the fluorescence intensity was changed from 13.8 to 123.5). However, the fluorescence signal of CMB and miRNA-21 was only 14.3 under the same conditions, which was almost the same as the CMB background signal (13.8). These data indicated that CMB could specifically recognize the pre-miR-21. To further verify the specificity of the CMB detection system, we tested the hybridization product by polyacrylamide gel electrophoresis. The staining result in Fig.2B showed that hybridization of CMB and pre-miR-21 produced a new band, while the original corresponding CMB band disappeared and leaving a small amount of pre-miR-21 in the lane 4. However, the sample containing CMB and miRNA-21 showed two bands (lane 5), and the size was exactly same as the individual CMB (lane 1) and miRNA-21 (lane 3). This result further indicated that the fluorescence signal produced by the hybridization of CMB and pre-miR-21. 3.4

Optimization of detection conditions

To optimize the condition of CMB detection system for pre-miR-21 assay, we investigated the effect of Mg2+ concentration[18], pH and time on the hybridization reaction. As shown in Fig.3A, the fluorescence background signal of CMB was relatively high without Mg2+. However, it became lower with an “L” shape upon the increase of Mg2+ from 0 to 30 mM. The results showed that the increase of Mg2+ concentration was helpful for the formation of hairpin structure, which inversely reduced the fluorescence background signal. Then, we investigated the effect of Mg2+ on the hybridization of CMB and pre-miR-21. As shown in the curve b of Fig.3A, the fluorescence signal of hybridization product showed an inverse “V” shape upon the increase of Mg2+ from 0 to 30 mM. The highest S/B ratio was obtained at 15 mM Mg2+ due to the low background and high hybrid signal. And then we studied the effect of pH on the hybridization. As demonstrated in the Fig.3B, the fluorescence background signal showed a “V” shape upon the change of pH from 5 to 9. The results showed that a low background signal and high hybridization signal at the assay were observed at pH 7.5. Next, we optimized the reactive time of reaction by comparing the fluorescence change at different points. The result showed that the fluorescence intensity increased as the extending of reaction time in the range of 5 to 60 min and it reached the maximum value after 50 min (the data not shown).

Fig.2 Feasibility analysis of CMB detection system. (A) Fluorescence spectra of CMB sensing system at different conditions; (B) Silver staining image of polyacrylamide gel electrophoresis results

Fig.3 Effect of concentration of Mg2+ and pH on hybridization reaction. (A) Relative fluorescence intensity of CMB detection system was quenched and recovered under different concentrationS of Mg2+. (B) Relative fluorescence intensity of CMB detection system was quenched and recovered under different pH value

XIE Hai-Long et al. / Chinese Journal of Analytical Chemistry, 2018, 46(7): e1847–e1853

3.5

Sensitivity of assay

To determine the potential use of the new established system for pre-miR-21 assay, we inspected the fluorescence restoration caused by target pre-miRNA in the NE buffer. As we expected, an enhancement of fluorescence intensity was observed after the addition of target pre-miRNA. A dramatic increase of fluorescence intensity was observed as the target concentration varied from 1 nM to 100 nM (Fig.4A). As shown in Fig.4B, the target concentration and the corresponding fluorescence value had a good linear relationship in the range of 1–100 nM with a corresponding coefficient (R2) of 0.979. Among of them, the regression equation was y = 0.442x + 13.745, where y represented the fluorescence intensities at 578 nm, x indicated the concentration of pre-miR-21. The detection limit (DL) was estimated as low as 0.5 nM (DL = 3.3 × (standard deviation/slope of the calibration curve)). Comparing with the sensitivity of previously reported miRNA detection sensors (Table 2), the new CMB sensor exhibited superior analytical performance for pre-miR-21 detection. 3.6

Application in complex biological samples

To examine the feasibility of CMB detection system for the pre-miR-21 assay in complex biological samples, we constructed MCF-7 cell lines with low expression of Z38 (sh35, sh146, sh227), over expression of Z38 (P.) and transfected with empty vector (con) as the detection object of this system[23]. Based on the fluorescence scan curves, we obtained the fluorescence signals from these samples at 578 nm. By combining with established standard curve, we calculated

the content of pre-miR-21 in different MCF-7 cell lines, as shown in Fig.5A and Table 3. The results displayed that the levels of pre-miR-21showed significant differences in treating MCF-7 cells. Among of them, the level of pre-miR-21 in the Z38 inhibitory group (sh35, sh146, sh227) was 3 times greater than the untreated group (ntc). However, the pre-miR-21 level did not show significant difference in the Z38 overexpressing cells (P.) and the bank vector-treating cells (control). We further verified the reliability of the CMB detection system for measuring pre-miR-21 using qRT-PCR method, and the results was indicated in Fig.5B. By comparing the results of these two methods, it was found that the pre-miR-21 level in different cell models was similar. 3.7

Expression detection of pre-miR-21 in tumor tissue by CMB

Previous studies have shown that pre-miR-21 levels are positively correlated with tumor malignancy[12]. We then examined the pre-miR-21 levels of clinical tumor samples using the CMB method. On the basis of the fluorescence signals from these samples at 578 nm, we calculated the concentration of pre-miR-21 according to the standard curve as shown in Fig.4B. The results in Fig.6A exhibited that the expression of pre-miR-21 gradually increased with the degree of malignancy from low to high in the glioma samples. Among of them, the clinical information of these patients was shown in the support information Table 1. We further investigated whether the new approach could discriminate the difference of pre-miR-21 in tumor and adjacent tissues. Sixteen pair gastric tissues were selected and the total RNAs extracted from these tissues were subjected to the fluorescence assay. As shown in

Table 2 Comparison of LOD level of different methods for miRNA assays Method

Complexity level

Molecular beacons Quantum dot-based FRET Hybrid chain reaction Rolling circle replication Chimeric molecular beacons

Easy Easy Complex Complex Easy

LOD

Ref.

1 nM 1 nM 7.4 fM 0.5 pM 0.5 nM

[19] [20] [21] [22] This study

Fig.4 Fluorescence emission spectra of different pre-miR-21 concentration. (A) Fluorescence spectra of CMB sensing system at a series of concentrations of pre-miR-21 (0‒100 nM). (B) The corresponding calibration plot of fluorescence intensity vs. the concentration of pre-miR-21 from 1 to 100 nM

XIE Hai-Long et al. / Chinese Journal of Analytical Chemistry, 2018, 46(7): e1847–e1853

Fig.5 (A) Quantitative detection of pre-miR-21 expression levels in different MCF-7 cell models. (B) Comparison of pre-miR-21 in different MCF-7 cell models by qRT-PCR and CMB assay. The error bars represent the standard deviation of three repetitive measurements

Fig.6 Detection of pre-miR-21 by CMB assay and qRT-PCR in different tumor tissue samples. (A, B) CMB method was used to detect the expression of pre-miR-21 in glioma tissues and gastric cancer tissues, respectively. (C, D) Analysis of variance about pre-miR-21 expression in two tumor tissue samples using CMB assay and qRT-PCR Table 3 Results of pre-miR-21 expression in different MCF-7 cell models Specimens

Pre-miR21 (×109 copy number μg–1 total RNA)

sh35 sh146 sh227 p. control ntc

2.145 1.867 1.755 1.017 0.898 0.663

Fig.6B, a different extent decrease of pre-miR-21 in the 12 patients (70%) compared with their corresponding adjacent tissues was observed. One patient did not show fluorescence signal change between the tumor and adjacent tissue. Meanwhile, the result indicated that pre-miR-21 level was related with the personal origin. Among of them, the clinical information of patients with gastric cancer was shown in the

support information Table 2. We further verified the reliability of the CMB detection system for measuring pre-miR-21 using qRT-PCR method. By comparing the results of the two methods in Fig.6C and Fig.6D, it was found that the pre-miR-21 level in different cell models was similar. Above results we can draw a conclusion that CMB assay will not only quantitatively detect the content of target molecules, but also reliably used for complex samples assay.

4

Conclusions

In summary, this study designed a simple, quantitative and specific method for pre-miR-21 assay using the chimeric molecular beacon composed of DNA loop and RNA stem. Based on this system, the purpose of sensitive and quantitative pre-miRNA detection was achieved, and the quantitative

XIE Hai-Long et al. / Chinese Journal of Analytical Chemistry, 2018, 46(7): e1847–e1853

detection was from 1 to 100 nM with the detection limit of 0.5 nM. In addition, the detection system could not only detect pre-miRNA in different tumor cell lines, but also was feasible for pre-miRNA detection in different originated tumor tissue samples. Based on these characteristics, this assay system would be hopeful to be used in pre-miRNA function study and prevention of tumors.

33(3): 275–279 [11] Deng R, Liu B, Wang Y, Feng Y, Hu S, Wang H, Wang T, Li B, Deng X, Xiang S. J. Cancer, 2016, 7(5): 576‒586 [12] Natsume A, Kinjo S, Yuki K, Kato T, Ohno M, Motomura K, Iwami K, Wakabayashi T. Brain Tumor Pathol., 2011, 28(1): 1–12 [13] Jiang Y X, Fang X H, Wan L J, Bai C L. Chinese J. Anal. Chem., 2004, 32(5): 668–672

References

[14] .Li C, Long Y, Liu B, Xiang D, Zhu H. Anal. Chim. Acta, 2014, 819: 71–77

[1]

Han J, Lee Y, Yeom K H, Nam J W, Heo I, Rhee J K, Sohn S Y, Cho Y, Zhang B T, Kim V N. Cell, 2006, 125(5): 887–901

[2]

Sun H L, Cui R, Zhou J, Teng K Y, Hsiao Y H, Nakanishi K, Fassan M, Luo Z, Shi G, Tili E. Cancer Cell, 2016, 30(5): 723–736

[3]

Bernstein E, Caudy A A, Hammond S M, Hannon G J. Nature, 2001, 409(6818): 363–366

[4]

Hutvágner G, McLachlan J, Pasquinelli A E, Bálint É, Tuschl T, Zamore P D. Science, 2001, 293(5531): 834–838

[5]

Li Z, Rana T M. Nat. Rev. Drug Discov., 2014, 13(8): 622–638

[6]

Lu Z, Liu M, Stribinskis V, Klinge C M, Ramos K S, Colburn

[7] [8]

Adams P D, Doudna J A. Science, 2006, 311(5758): 195‒198 [16] Ke G, Wang C, Ge Y, Zheng N, Zhu Z, Yang C J. J. Am. Chem. Soc., 2012, 134(46): 18908–18911 [17] Wang L, Yang C J, Medley C D, Benner S A, Tan W H. J. Am. Chem. Soc., 2005, 127(45): 15664–15665 [18] Fan J L, Zhang X Z, Cheng Y X, Xiao C H, Wang W, Liu X M, Tong C Y, Liu B. RSC Adv., 2017, 7(57): 35629–35637 [19] Baker M B, Bao G, Searles C D. Nucleic Acids Res., 2011, 40(2): e13

N H, Li Y. Oncogene, 2008, 27(31): 4373–4379

[20] Qiu X, Hildebrandt N. ACS Nano, 2015, 9(8): 8449–8457

Pall G S, Hamilton A J. Nat. Protocols, 2008, 3(6): 1077–1084

[21] Wu H, Liu Y, Wang H, Wu J, Zhu F, Zou P. Biosens.

Tong L, Xue H, Xiong L, Xiao J, Zhou Y. Mol. Biotechnol., 2015, 5 (10): 939–946

[9]

[15] Macrae I J, Zhou K, Li F, Repic A, Brooks A N, Cande W Z,

James A M, Baker M B, Bao G, Searles C D. Theranostics, 2017, 7(3): 634

[10] Xiang D, Zhai K, Sang Q, Shi B, Yang X. Anal. Sci., 2017,

Bioelectron., 2016, 81: 303–308 [22] Tang Y, Wang T, Chen M, He X, Qu X, Feng X. Biosens. Bioelectron., 2016, 85: 151–156 [23] Deng R, Liu B, Wang Y, Yan F, Hu S, Wang H, Wang T, Li B, Deng X, Xiang S. J. Cancer, 2016, 7(5): 576–586