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Surface-enhanced Raman spectroscopy for simultaneous sensitive detection of multiple microRNAs in lung cancer cells†
Received 20th July 2014, Accepted 14th August 2014
Li-Ping Ye,‡a Juan Hu,‡a Li Liang‡b and Chun-yang Zhang*a
DOI: 10.1039/c4cc05598e www.rsc.org/chemcomm
We develop circular exponential amplification reaction (EXPAR)-based surface-enhanced Raman spectroscopy (SERS) for simultaneous sensitive detection of multiple microRNAs in non-small cell lung cancer cells. This method possesses distinct advantages of excellent selectivity and high sensitivity with a detection limit of as low as 0.5 fM, which has improved by as much as 6 orders of magnitude as compared with the previously reported SERS-based direct assay.
The microRNAs (miRNAs) are a class of evolutionally conserved, small non-coding RNAs.1 The miRNAs play essential roles in posttranscriptional regulation of genes and involve in a range of crucial biological processes including cell proliferation, differentiation, apoptosis and immune systems.1,2 The dysregulation of miRNAs is associated with many pathological processes such as metabolic disorders and the altered immune system function.3 Some miRNAs actually function as oncogenes or tumor suppressors in cancers.1a,4 Globally, lung cancer is the most mortal cancer that comprises small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Almost 80% of lung cancer is diagnosed to be NSCLC which includes squamous cell lung carcinoma (SCC), lung adenocarcinoma (LAD) and large cell lung cancer.5 Recent research reveals the aberrant expression of some tumor-specific miRNAs such as miR-205 and miR-126 in lung cancer.6 MiR-205 is expressed at a higher level in squamous cell lung carcinoma than in nonsquamous NSCLC,7 and miR-126 as a potential tumor suppressor is down-regulated in lung cancers.8 Therefore, the measurement of aberrant expression of miR-205 and miR-126 might be useful for the clinical diagnosis of lung cancer. The miRNAs have unique characteristics of small size, low abundance and sequence homology, making their accurate a
Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong 518055, China. E-mail: [email protected]; Fax: +86-755-86392299; Tel: +86-755-86392211 b Department of Tumor Chemotherapy and Radiation Sickness, Peking University Third Hospital, Beijing 100191, China † Electronic supplementary information (ESI) available: Details of experimental procedures and additional figures. See DOI: 10.1039/c4cc05598e ‡ These authors contributed equally.
This journal is © The Royal Society of Chemistry 2014
detection remain a great challenge. Northern blotting is widely used for miRNA assay,9 but it is time-consuming with the requirement of a large amount of total RNA, and often fails to detect low-abundant miRNA. Microarray has the advantage of high throughput but it tends to have low specificity and a small dynamic range, while RNA sequencing is too expensive to be widely accessible.10 The PCR-based method relies on the reverse transcription of miRNA to cDNA, and is restricted by the short length of miRNAs.10,11 Alternatively, some new approaches based on organic fluorophores,12 graphene oxide13 and quantum dots14 have been developed to improve the detection sensitivity and specificity. Recently, isothermal nucleic acid amplification is emerging as a promising technique for molecular diagnostics.15 The isothermal exponential amplification reaction (EXPAR) can generate abundant short oligonucleotides with a high amplification efficiency of 4106-fold within minutes.16 In the classical EXPAR assay,15a SYBR Green I is usually used as the fluorescent indicator, but the SYBR Green I dye has some limitations such as the inhibition of PCR reaction, the promotion of nonspecific amplification, and being unable to distinguish multiple targets.17 To improve both the detection specificity and the multiplexed capability, we develop exponential amplification reaction (EXPAR)-based surface-enhanced Raman spectroscopy (SERS) for simultaneous sensitive detection of multiple microRNAs in NSCLC. SERS is characterized by its capability to identify target analytes with an information-rich vibrational spectrum, and its narrow well-resolved peaks allow for simultaneous detection of multiple targets.18 SERS has been widely used for the detection of nucleic acids,19 proteins,20 glucoses,21 drugs,22 even mammalian cells and tissues.23 Even though miRNAs can be directly identified by SERS,24 the structural similarities of oligonucleotides might result in the overlapping peaks that are difficult to be differentiated. In EXPAR-based SERS, the EXPAR reaction can produce numerous short single-stranded oligonucleotides, which can specifically bind with the reporter probes and the AuNP-modified capture probes to form the sandwich hybrids, resulting in an enhanced SERS signal. The principle of EXPAR-based SERS for miRNA assay is shown in Scheme 1. The template of EXPAR contains a sequence of X,
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Fig. 1 SERS spectra (A) and variance of Raman intensity (B) as a function of miR-205 concentration. The Raman intensity is quantitatively analyzed by the measurement of Raman intensity at 1651 cm 1. Error bars show the standard deviation of three experiments.
Scheme 1
Schematic illustration of EXPAR-based SERS for microRNA assay.
which was complementary to the target miRNA, and two repeat sequences of T–T. EXPAR involves two reactions as follows: (1) a linear amplification enabling the conversion of miRNA to the trigger; (2) a circular exponential amplification enabling the continuous production of abundant triggers. In the presence of target miRNA, the annealing of miRNA with the sequence X of the template initiates the linear amplification. The resultant partial duplex is extended in the presence of Vent (exo-) DNA polymerase to obtain a double-stranded DNA (dsDNA) duplex with two recognition sites for the nicking endonuclease Nt.BstNBI. Followed by the cleavage of the upper DNA strand from dsDNA by Nt.BstNBI, Vent (exo-) DNA polymerase initiates a new round of replication at the nick to displace the cleaved trigger. At the mean time, new recognition sites are formed to make the above reaction proceed repeatedly. As a result, a small amount of miRNAs can be transferred to abundant triggers through this linear amplification. Furthermore, the released trigger might function as a primer by annealing the first sequence T of the template to initiate a circular exponential amplification. Consequently, large amounts of short single-stranded triggers can be obtained through the repeated extension, cleavage and release. The resultant single-stranded triggers can be further sandwiched by a reporter probe and a AuNP-modified capture probe25 to obtain a sandwiched hybrid. Due to the presence of abundant capture probes on the surface of AuNP, large amounts of reporter probes can be absorbed on the surface of single AuNP, thus significantly enhancing the SERS signal and enabling the sensitive detection of lowabundance miRNAs. The target miRNA-induced EXPAR reaction is confirmed by real time fluorescence measurement as well as PAGE analysis (see ESI,† Fig. S1A and B). As shown in Fig. S1C (ESI†), in the presence of miR-205, template, polymerase and nicking enzyme, a strong characteristic SERS signal is observed. In contrast, no significant SERS signal is observed in the absence of miR-205 despite some weak SERS signals due to the nonspecific adsorption of reporter probes on the surface of the AuNPs.19b,26 These results demonstrate that EXPAR-based SERS can be applied for sensitive detection of miR-205.
11884 | Chem. Commun., 2014, 50, 11883--11886
We further investigated the sensitivity of EXPAR-based SERS under the optimum conditions (see ESI,† Fig. S2). The target miR-205 initiates the EXPAR reaction and produces large amounts of triggers, which can subsequently hybridize with TAMRA-labeled reporter probes and AuNP-modified capture probes (see ESI,† Fig. S3) to form the sandwich hybrids. The obtained sandwich hybrids can generate characteristic SERS spectra at an excitation wavelength of 632.8 nm. As shown in Fig. 1A, the SERS signal increases with the concentration of target miR-205 from 1 fM to 100 nM. By measuring the Raman intensity at 1651 cm 1, we quantitatively analyzed the variance of Raman intensity with the concentration of target miRNA (Fig. 1B). The Raman intensity shows a good linear fit to the logarithm of target miRNA in the range from 1.0 10 15 M to 1.0 10 7 M. The correlation equation is I = 252.3log10 C + 4087.8, where I and C are the Raman intensity and the concentration of target miRNA (M), respectively, and the correlation coefficient is 0.994. The detection limit is estimated to be 0.5 fM by calculating the average response of the blank plus three times the standard deviation. Notably, the sensitivity of EXPAR-based SERS has improved by as much as 8 orders of magnitude as compared with the label-free SERS method (28 nM)24b and 6 orders of magnitude as compared with the previously reported SERS-based direct assay (0.36 nM).27 The improved sensitivity of EXPAR-based SERS might be attributed to the extremely high amplification efficiency of circular EXPAR (4106-fold)16 and the high signal enhancement of SERS.18a Moreover, this method exhibits good reproducibility (see ESI,† Fig. S4). Specific detection of miRNA remains a great challenge because different miRNAs often possess closely related sequences with a few bases difference. According to the latest miRBase Sequence Database, miR-7157 has the most similar sequence to miR-205 in all human miRNAs (see ESI,† Table S1). In this research, we employed miR-205, one-base mismatched miRNA and miR-7157 to investigate the detection specificity of EXPAR-based SERS (see ESI,† Fig. S5). With the miR-205-specific template, the SERS signal in response to miR-205, one-base mismatched miRNA and miR7157 is 13.5-, 5.0-, and 1.9-fold higher than that obtained from the control group without target miRNA, respectively. These results demonstrate that EXPAR-based SERS can distinguish the perfectmatched miRNA from those with a one-base mismatched and similar sequence. With the capability of identifying the molecular fingerprint information,19a EXPAR-based SERS can be further applied for
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multiplexed miRNA assay. To demonstrate the principle of concept, we designed two specific templates for miR-126 and miR-205, respectively, to generate the specific triggers through EXPAR. The miR-126-induced trigger can hybridize with the Cy3-labeled reporter probe and the AuNP-modified capture probe to form the Cy3-labeled sandwich hybrids. Similarly, the miR-205-induced trigger can hybridize with the TAMRAlabeled reporter probe and the AuNP-modified capture probe to form the TAMRA-labeled sandwich hybrids. The absorption of abundant specific dye-labeled sandwich hybrids on the surface of single AuNP can generate characteristic SERS signals with the molecular fingerprint information. The Cy3-labeled reporter probes can produce a characteristic SERS signal at 1590 cm 1, indicating the presence of target miR-126, while the TAMRA-labeled reporter probes can produce a characteristic SERS signal at 1651 cm 1, indicating the presence of target miR-205. Notably, the same capture probe is employed for miR-126 and miR-205 (see ESI,† Table S1), leading to the assembly of both Cy3-labeled and TAMRA-labeled reporter probes on the surface of single AuNP in the presence of both miR-126 and miR-205, and consequently the characteristic SERS signals at 1590 cm 1 and 1651 cm 1 can be observed simultaneously (Fig. 2A). Fig. 2B shows the quantitative results. miR-126 induces high Raman intensity at 1590 cm 1 instead of at 1651 cm 1, while miR-205 induces high Raman intensity at 1651 cm 1 instead of at 1590 cm 1. Only the co-existence of miR-126 and miR-205 can induce high Raman intensities at both 1590 cm 1 and 1651 cm 1. These results demonstrate the feasibility of EXPAR-based SERS for multiplexed miRNA assay. To investigate the capability of EXPAR-based SERS for real sample analysis, we further examine the expression of miR-126 and miR-205 in Beas-2B cells (immortalized human bronchial epithelial cell lines) and NSCLC cell lines A549 (adenocarcinoma), NCI-H596 (H596, adenosquamous carcinoma), SK-MES-1 (squamous cell lung carcinoma). We measured the miRNAs extracted from the above cell lines using two specific templates for miR-126 and miR-205, respectively. Based on the measured Raman intensity at 1590 cm 1 and 1651 cm 1 (Fig. 3), there are a lower level of miR-126 and a higher level of miR-205 in A549, H596, SK-MES-1 cells than in Bear-2B cells, suggesting that the expression of miR-126 is downregulated while the expression
Fig. 2 Multiplexed miRNA assay. (A) Raman spectra of amplification products in the presence of miR-126 (blue line), miR-205 (red line), and a mixture of miR-126 and miR-205 (black line), respectively. The concentration of miR-126 is 1 pM, and the concentration of miR-205 is 1 pM. (B) Simultaneous detection of miR-126 (green column) and miR-205 (purple column) by measuring Raman intensity at 1590 cm 1 for miR-126 and 1651 cm 1 for miR-205. Error bars show the standard deviation of three experiments.
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Fig. 3 Expression of miR-126 (green column) and miR-205 (purple column) in Beas-2B, A549, H596 and SK-MES-1 cells analyzed by Raman intensity at 1590 cm 1 and 1651 cm 1. Error bars show the standard deviations of three experiments.
of miR-205 is upregulated in NSCLC cell lines, consistent with the previous research.6–8 In addition, the expression of miR-205 increases in the order of A549, H596 and SK-MES-1 cells, suggesting the higher expression of miR-205 in squamous cell lung carcinoma than in nonsquamous cell lung carcinoma.7 These results demonstrate that EXPAR-based SERS can be used to sensitively detect miRNAs in the cells. We further compared EXPAR-based SERS with qPCR for miR-205 and miR-126 assay in the clinical tissue samples. The results obtained by EXPAR-based SERS agree well with those obtained by qPCR (see ESI,† Fig. S6–S8). Notably, the miR-126 concentration obtained from the healthy persons is significantly higher than that obtained from the squamous cell lung carcinoma patients (unpaired t test, P = 0.0077, see ESI,† Fig. S7), while the miR-205 concentration obtained from the squamous cell lung carcinoma patients is significantly higher than that obtained from the healthy persons (unpaired t test, P = 0.0003, see ESI,† Fig. S7). In addition, we employed EXPAR-based SERS to measure the tissue samples with the spiked miR-205. The obtained recovery rate is in the range of 90.52–94.31% (see ESI,† Table S2). These results demonstrate that EXPAR-based SERS holds great promise for miRNA assay with great accuracy and reliability. In conclusion, we have developed an EXPAR-based SERS method for simultaneous sensitive detection of multiple miRNAs in lung cancers. Owing to the high amplification efficiency of circular EXPAR and the high signal enhancement of SERS, this method possesses distinct advantages of high sensitivity and excellent selectivity. The detection limit of EXPAR-based SERS can reach as low as 0.5 fM, which has improved by as much as 8 orders of magnitude as compared with the label-free SERS method24b and 6 orders of magnitude as compared with the previously reported SERS-based direct assay,27 and 3 orders of magnitude as compared with the EXPAR-based electrochemical biosensor.28 EXPAR-based SERS may discriminate target miRNA from the miRNAs with high sequence homology, and can be used to simultaneously detect multiple microRNAs in lung cancer cells, having great potential for further application in early clinical diagnosis. This work was supported by the National Natural Science Foundation of China (Grant No. 21325523 and 21205128), the Award for the Hundred Talent Program of the Chinese Academy of Sciences, the Fund for Shenzhen Engineering Laboratory of
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Single-molecule Detection and Instrument Development (Grant No. (2012) 433), the funds from State Ministry of Health of China (Grant No. 201002009) and Peking University Third Hospital (Grant No. 2013-BYSY-CAS-007).
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Notes and references 1 (a) R. Schickel, B. Boyerinas, S. M. Park and M. E. Peter, Oncogene, 2008, 27, 5959; (b) J. Krol, I. Loedige and W. Filipowicz, Nat. Rev. Genet., 2010, 11, 597. 2 V. Ambros, Curr. Opin. Genet. Dev., 2011, 21, 511. 3 (a) A. Zampetaki, S. Kiechl, I. Drozdov, P. Willeit, U. Mayr, M. Prokopi, A. Mayr, S. Weger, F. Oberhollenzer, E. Bonora, A. Shah, J. Willeit and M. Mayr, Circ. Res., 2010, 107, 810; (b) T. X. Lu and M. E. Rothenberg, J. Allergy Clin. Immunol., 2013, 132, 3. 4 S. Babashah and M. Soleimani, Eur. J. Cancer, 2011, 47, 1127. 5 C. H. Zhou, L. P. Ye, S. X. Ye, Y. Li, X. Y. Zhang, X. Y. Xu and L. Y. Gong, J. Exp. Clin. Cancer Res., 2012, 31, 18. 6 U. Vosa, T. Vooder, R. Kolde, J. Vilo, A. Metspalu and T. Annilo, Int. J. Cancer, 2013, 132, 2884. 7 (a) D. Lebanony, H. Benjamin, S. Gilad, M. Ezagouri, A. Dov, K. Ashkenazi, N. Gefen, S. Izraeli, G. Rechavi, H. Pass, D. Nonaka, J. Li, Y. Spector, N. Rosenfeld, A. Chajut, D. Cohen, R. Aharonov and M. Mansukhani, J. Clin. Oncol., 2009, 27, 2030; (b) J. Cai, L. Fang, Y. Huang, R. Li, J. Yuan, Y. Yang, X. Zhu, B. Chen, J. Wu and M. Li, Cancer Res., 2013, 73, 5402. 8 B. Liu, X. C. Peng, X. L. Zheng, J. Wang and Y. W. Qin, Lung Cancer, 2009, 66, 169. 9 A. G. Torres, M. M. Fabani, E. Vigorito and M. J. Gait, RNA, 2011, 17, 933. 10 C. C. Pritchard, H. H. Cheng and M. Tewari, Nat. Rev. Genet., 2012, 13, 358. 11 N. Redshaw, T. Wilkes, A. Whale, S. Cowen, J. Huggett and C. A. Foy, Biotechniques, 2013, 54, 155. 12 R. Duan, X. Zuo, S. Wang, X. Quan, D. Chen, Z. Chen, L. Jiang, C. Fan and F. Xia, J. Am. Chem. Soc., 2013, 135, 4604. 13 C. Liu, Z. Wang, H. Jia and Z. Li, Chem. Commun., 2011, 47, 4661. 14 Y. Zhang and C. Y. Zhang, Anal. Chem., 2012, 84, 224. 15 (a) H. Jia, Z. Li, C. Liu and Y. Cheng, Angew. Chem., Int. Ed., 2010, 49, 5498; (b) F. Wang, L. Freage, R. Orbach and I. Willner, Anal.
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Chem., 2013, 85, 8196; (c) F. Wang, C. H. Lu, X. Liu, L. Freage and I. Willner, Anal. Chem., 2014, 86, 1614; (d) Z. Z. Zhang and C. Y. Zhang, Anal. Chem., 2012, 84, 1623. J. Van Ness, L. K. Van Ness and D. J. Galas, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 4504. H. Gudnason, M. Dufva, D. D. Bang and A. Wolff, Nucleic Acids Res., 2007, 35, e127. (a) M. D. Sonntag, J. M. Klingsporn, A. B. Zrimsek, B. Sharma, L. K. Ruvuna and R. P. Van Duyne, Chem. Soc. Rev., 2014, 43, 1230; (b) X. X. Han, B. Zhao and Y. Ozaki, TrAC, Trends Anal. Chem., 2012, 38, 67; (c) X. M. Qian, X. H. Peng, D. O. Ansari, Q. Yin-Goen, G. Z. Chen, D. M. Shin, L. Yang, A. N. Young, M. D. Wang and S. M. Nie, Nat. Biotechnol., 2008, 26, 83. (a) Y. C. Cao, R. Jin and C. A. Mirkin, Science, 2002, 297, 1536; (b) J. Hu and C. Y. Zhang, Anal. Chem., 2010, 82, 8991; (c) H. T. Ngo, H. N. Wang, A. M. Fales and T. Vo-Dinh, Anal. Chem., 2013, 85, 6378; (d) L. Fabris, M. Dante, G. Braun, S. J. Lee, N. O. Reich, M. Moskovits, T. Q. Nguyen and G. C. Bazan, J. Am. Chem. Soc., 2007, 129, 6086; (e) L. Sun, C. Yu and J. Irudayaraj, Anal. Chem., 2007, 79, 3981. (a) J. Yan, S. Su, S. He, Y. He, B. Zhao, D. Wang, H. Zhang, Q. Huang, S. Song and C. Fan, Anal. Chem., 2012, 84, 9139; (b) L. Lesser-Rojas, P. Ebbinghaus, G. Vasan, M. L. Chu, A. Erbe and C. F. Chou, Nano Lett., 2014, 14, 2242. J. M. Yuen, N. C. Shah, J. J. Walsh, M. R. Glucksberg and R. P. Van Duyne, Anal. Chem., 2010, 82, 8382. C. S. Levin, J. Kundu, B. G. Janesko, G. E. Scuseria, R. M. Raphael and N. J. Halas, J. Phys. Chem. B, 2008, 112, 14168. (a) K. V. Kong, Z. Lam, W. D. Goh, W. K. Leong and M. Olivo, Angew. Chem., Int. Ed., 2012, 51, 9796; (b) M. Salehi, L. Schneider, P. Strobel, A. Marx, J. Packeisen and S. Schlucker, Nanoscale, 2014, 6, 2361. (a) J. D. Driskell, A. G. Seto, L. P. Jones, S. Jokela, R. A. Dluhy, Y. P. Zhao and R. A. Tripp, Biosens. Bioelectron., 2008, 24, 917; (b) J. D. Driskell and R. A. Tripp, Chem. Commun., 2010, 46, 3298. C. A. Mirkin, R. L. Letsinger, R. C. Mucic and J. J. Storhoff, Nature, 1996, 382, 607. G. Wang, R. J. Lipert, M. Jain, S. Kaur, S. Chakraboty, M. P. Torres, S. K. Batra, R. E. Brand and M. D. Porter, Anal. Chem., 2011, 83, 2554. B. Guven, F. C. Dudak, I. H. Boyaci, U. Tamer and M. Ozsoz, Analyst, 2014, 139, 1141. Y. R. Yan, D. Zhao, T. X. Yuan, J. Hu, D. C. Zhang, W. Cheng, W. Zhang and S. J. Ding, Electroanalysis, 2013, 25, 2354.
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Cite this: Chem. Commun., 2014, 50, 11883
Surface-enhanced Raman spectroscopy for simultaneous sensitive detection of multiple microRNAs in lung cancer cells†
Received 20th July 2014, Accepted 14th August 2014
Li-Ping Ye,‡a Juan Hu,‡a Li Liang‡b and Chun-yang Zhang*a
DOI: 10.1039/c4cc05598e www.rsc.org/chemcomm
We develop circular exponential amplification reaction (EXPAR)-based surface-enhanced Raman spectroscopy (SERS) for simultaneous sensitive detection of multiple microRNAs in non-small cell lung cancer cells. This method possesses distinct advantages of excellent selectivity and high sensitivity with a detection limit of as low as 0.5 fM, which has improved by as much as 6 orders of magnitude as compared with the previously reported SERS-based direct assay.
The microRNAs (miRNAs) are a class of evolutionally conserved, small non-coding RNAs.1 The miRNAs play essential roles in posttranscriptional regulation of genes and involve in a range of crucial biological processes including cell proliferation, differentiation, apoptosis and immune systems.1,2 The dysregulation of miRNAs is associated with many pathological processes such as metabolic disorders and the altered immune system function.3 Some miRNAs actually function as oncogenes or tumor suppressors in cancers.1a,4 Globally, lung cancer is the most mortal cancer that comprises small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Almost 80% of lung cancer is diagnosed to be NSCLC which includes squamous cell lung carcinoma (SCC), lung adenocarcinoma (LAD) and large cell lung cancer.5 Recent research reveals the aberrant expression of some tumor-specific miRNAs such as miR-205 and miR-126 in lung cancer.6 MiR-205 is expressed at a higher level in squamous cell lung carcinoma than in nonsquamous NSCLC,7 and miR-126 as a potential tumor suppressor is down-regulated in lung cancers.8 Therefore, the measurement of aberrant expression of miR-205 and miR-126 might be useful for the clinical diagnosis of lung cancer. The miRNAs have unique characteristics of small size, low abundance and sequence homology, making their accurate a
Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong 518055, China. E-mail: [email protected]; Fax: +86-755-86392299; Tel: +86-755-86392211 b Department of Tumor Chemotherapy and Radiation Sickness, Peking University Third Hospital, Beijing 100191, China † Electronic supplementary information (ESI) available: Details of experimental procedures and additional figures. See DOI: 10.1039/c4cc05598e ‡ These authors contributed equally.
This journal is © The Royal Society of Chemistry 2014
detection remain a great challenge. Northern blotting is widely used for miRNA assay,9 but it is time-consuming with the requirement of a large amount of total RNA, and often fails to detect low-abundant miRNA. Microarray has the advantage of high throughput but it tends to have low specificity and a small dynamic range, while RNA sequencing is too expensive to be widely accessible.10 The PCR-based method relies on the reverse transcription of miRNA to cDNA, and is restricted by the short length of miRNAs.10,11 Alternatively, some new approaches based on organic fluorophores,12 graphene oxide13 and quantum dots14 have been developed to improve the detection sensitivity and specificity. Recently, isothermal nucleic acid amplification is emerging as a promising technique for molecular diagnostics.15 The isothermal exponential amplification reaction (EXPAR) can generate abundant short oligonucleotides with a high amplification efficiency of 4106-fold within minutes.16 In the classical EXPAR assay,15a SYBR Green I is usually used as the fluorescent indicator, but the SYBR Green I dye has some limitations such as the inhibition of PCR reaction, the promotion of nonspecific amplification, and being unable to distinguish multiple targets.17 To improve both the detection specificity and the multiplexed capability, we develop exponential amplification reaction (EXPAR)-based surface-enhanced Raman spectroscopy (SERS) for simultaneous sensitive detection of multiple microRNAs in NSCLC. SERS is characterized by its capability to identify target analytes with an information-rich vibrational spectrum, and its narrow well-resolved peaks allow for simultaneous detection of multiple targets.18 SERS has been widely used for the detection of nucleic acids,19 proteins,20 glucoses,21 drugs,22 even mammalian cells and tissues.23 Even though miRNAs can be directly identified by SERS,24 the structural similarities of oligonucleotides might result in the overlapping peaks that are difficult to be differentiated. In EXPAR-based SERS, the EXPAR reaction can produce numerous short single-stranded oligonucleotides, which can specifically bind with the reporter probes and the AuNP-modified capture probes to form the sandwich hybrids, resulting in an enhanced SERS signal. The principle of EXPAR-based SERS for miRNA assay is shown in Scheme 1. The template of EXPAR contains a sequence of X,
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Fig. 1 SERS spectra (A) and variance of Raman intensity (B) as a function of miR-205 concentration. The Raman intensity is quantitatively analyzed by the measurement of Raman intensity at 1651 cm 1. Error bars show the standard deviation of three experiments.
Scheme 1
Schematic illustration of EXPAR-based SERS for microRNA assay.
which was complementary to the target miRNA, and two repeat sequences of T–T. EXPAR involves two reactions as follows: (1) a linear amplification enabling the conversion of miRNA to the trigger; (2) a circular exponential amplification enabling the continuous production of abundant triggers. In the presence of target miRNA, the annealing of miRNA with the sequence X of the template initiates the linear amplification. The resultant partial duplex is extended in the presence of Vent (exo-) DNA polymerase to obtain a double-stranded DNA (dsDNA) duplex with two recognition sites for the nicking endonuclease Nt.BstNBI. Followed by the cleavage of the upper DNA strand from dsDNA by Nt.BstNBI, Vent (exo-) DNA polymerase initiates a new round of replication at the nick to displace the cleaved trigger. At the mean time, new recognition sites are formed to make the above reaction proceed repeatedly. As a result, a small amount of miRNAs can be transferred to abundant triggers through this linear amplification. Furthermore, the released trigger might function as a primer by annealing the first sequence T of the template to initiate a circular exponential amplification. Consequently, large amounts of short single-stranded triggers can be obtained through the repeated extension, cleavage and release. The resultant single-stranded triggers can be further sandwiched by a reporter probe and a AuNP-modified capture probe25 to obtain a sandwiched hybrid. Due to the presence of abundant capture probes on the surface of AuNP, large amounts of reporter probes can be absorbed on the surface of single AuNP, thus significantly enhancing the SERS signal and enabling the sensitive detection of lowabundance miRNAs. The target miRNA-induced EXPAR reaction is confirmed by real time fluorescence measurement as well as PAGE analysis (see ESI,† Fig. S1A and B). As shown in Fig. S1C (ESI†), in the presence of miR-205, template, polymerase and nicking enzyme, a strong characteristic SERS signal is observed. In contrast, no significant SERS signal is observed in the absence of miR-205 despite some weak SERS signals due to the nonspecific adsorption of reporter probes on the surface of the AuNPs.19b,26 These results demonstrate that EXPAR-based SERS can be applied for sensitive detection of miR-205.
11884 | Chem. Commun., 2014, 50, 11883--11886
We further investigated the sensitivity of EXPAR-based SERS under the optimum conditions (see ESI,† Fig. S2). The target miR-205 initiates the EXPAR reaction and produces large amounts of triggers, which can subsequently hybridize with TAMRA-labeled reporter probes and AuNP-modified capture probes (see ESI,† Fig. S3) to form the sandwich hybrids. The obtained sandwich hybrids can generate characteristic SERS spectra at an excitation wavelength of 632.8 nm. As shown in Fig. 1A, the SERS signal increases with the concentration of target miR-205 from 1 fM to 100 nM. By measuring the Raman intensity at 1651 cm 1, we quantitatively analyzed the variance of Raman intensity with the concentration of target miRNA (Fig. 1B). The Raman intensity shows a good linear fit to the logarithm of target miRNA in the range from 1.0 10 15 M to 1.0 10 7 M. The correlation equation is I = 252.3log10 C + 4087.8, where I and C are the Raman intensity and the concentration of target miRNA (M), respectively, and the correlation coefficient is 0.994. The detection limit is estimated to be 0.5 fM by calculating the average response of the blank plus three times the standard deviation. Notably, the sensitivity of EXPAR-based SERS has improved by as much as 8 orders of magnitude as compared with the label-free SERS method (28 nM)24b and 6 orders of magnitude as compared with the previously reported SERS-based direct assay (0.36 nM).27 The improved sensitivity of EXPAR-based SERS might be attributed to the extremely high amplification efficiency of circular EXPAR (4106-fold)16 and the high signal enhancement of SERS.18a Moreover, this method exhibits good reproducibility (see ESI,† Fig. S4). Specific detection of miRNA remains a great challenge because different miRNAs often possess closely related sequences with a few bases difference. According to the latest miRBase Sequence Database, miR-7157 has the most similar sequence to miR-205 in all human miRNAs (see ESI,† Table S1). In this research, we employed miR-205, one-base mismatched miRNA and miR-7157 to investigate the detection specificity of EXPAR-based SERS (see ESI,† Fig. S5). With the miR-205-specific template, the SERS signal in response to miR-205, one-base mismatched miRNA and miR7157 is 13.5-, 5.0-, and 1.9-fold higher than that obtained from the control group without target miRNA, respectively. These results demonstrate that EXPAR-based SERS can distinguish the perfectmatched miRNA from those with a one-base mismatched and similar sequence. With the capability of identifying the molecular fingerprint information,19a EXPAR-based SERS can be further applied for
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multiplexed miRNA assay. To demonstrate the principle of concept, we designed two specific templates for miR-126 and miR-205, respectively, to generate the specific triggers through EXPAR. The miR-126-induced trigger can hybridize with the Cy3-labeled reporter probe and the AuNP-modified capture probe to form the Cy3-labeled sandwich hybrids. Similarly, the miR-205-induced trigger can hybridize with the TAMRAlabeled reporter probe and the AuNP-modified capture probe to form the TAMRA-labeled sandwich hybrids. The absorption of abundant specific dye-labeled sandwich hybrids on the surface of single AuNP can generate characteristic SERS signals with the molecular fingerprint information. The Cy3-labeled reporter probes can produce a characteristic SERS signal at 1590 cm 1, indicating the presence of target miR-126, while the TAMRA-labeled reporter probes can produce a characteristic SERS signal at 1651 cm 1, indicating the presence of target miR-205. Notably, the same capture probe is employed for miR-126 and miR-205 (see ESI,† Table S1), leading to the assembly of both Cy3-labeled and TAMRA-labeled reporter probes on the surface of single AuNP in the presence of both miR-126 and miR-205, and consequently the characteristic SERS signals at 1590 cm 1 and 1651 cm 1 can be observed simultaneously (Fig. 2A). Fig. 2B shows the quantitative results. miR-126 induces high Raman intensity at 1590 cm 1 instead of at 1651 cm 1, while miR-205 induces high Raman intensity at 1651 cm 1 instead of at 1590 cm 1. Only the co-existence of miR-126 and miR-205 can induce high Raman intensities at both 1590 cm 1 and 1651 cm 1. These results demonstrate the feasibility of EXPAR-based SERS for multiplexed miRNA assay. To investigate the capability of EXPAR-based SERS for real sample analysis, we further examine the expression of miR-126 and miR-205 in Beas-2B cells (immortalized human bronchial epithelial cell lines) and NSCLC cell lines A549 (adenocarcinoma), NCI-H596 (H596, adenosquamous carcinoma), SK-MES-1 (squamous cell lung carcinoma). We measured the miRNAs extracted from the above cell lines using two specific templates for miR-126 and miR-205, respectively. Based on the measured Raman intensity at 1590 cm 1 and 1651 cm 1 (Fig. 3), there are a lower level of miR-126 and a higher level of miR-205 in A549, H596, SK-MES-1 cells than in Bear-2B cells, suggesting that the expression of miR-126 is downregulated while the expression
Fig. 2 Multiplexed miRNA assay. (A) Raman spectra of amplification products in the presence of miR-126 (blue line), miR-205 (red line), and a mixture of miR-126 and miR-205 (black line), respectively. The concentration of miR-126 is 1 pM, and the concentration of miR-205 is 1 pM. (B) Simultaneous detection of miR-126 (green column) and miR-205 (purple column) by measuring Raman intensity at 1590 cm 1 for miR-126 and 1651 cm 1 for miR-205. Error bars show the standard deviation of three experiments.
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Fig. 3 Expression of miR-126 (green column) and miR-205 (purple column) in Beas-2B, A549, H596 and SK-MES-1 cells analyzed by Raman intensity at 1590 cm 1 and 1651 cm 1. Error bars show the standard deviations of three experiments.
of miR-205 is upregulated in NSCLC cell lines, consistent with the previous research.6–8 In addition, the expression of miR-205 increases in the order of A549, H596 and SK-MES-1 cells, suggesting the higher expression of miR-205 in squamous cell lung carcinoma than in nonsquamous cell lung carcinoma.7 These results demonstrate that EXPAR-based SERS can be used to sensitively detect miRNAs in the cells. We further compared EXPAR-based SERS with qPCR for miR-205 and miR-126 assay in the clinical tissue samples. The results obtained by EXPAR-based SERS agree well with those obtained by qPCR (see ESI,† Fig. S6–S8). Notably, the miR-126 concentration obtained from the healthy persons is significantly higher than that obtained from the squamous cell lung carcinoma patients (unpaired t test, P = 0.0077, see ESI,† Fig. S7), while the miR-205 concentration obtained from the squamous cell lung carcinoma patients is significantly higher than that obtained from the healthy persons (unpaired t test, P = 0.0003, see ESI,† Fig. S7). In addition, we employed EXPAR-based SERS to measure the tissue samples with the spiked miR-205. The obtained recovery rate is in the range of 90.52–94.31% (see ESI,† Table S2). These results demonstrate that EXPAR-based SERS holds great promise for miRNA assay with great accuracy and reliability. In conclusion, we have developed an EXPAR-based SERS method for simultaneous sensitive detection of multiple miRNAs in lung cancers. Owing to the high amplification efficiency of circular EXPAR and the high signal enhancement of SERS, this method possesses distinct advantages of high sensitivity and excellent selectivity. The detection limit of EXPAR-based SERS can reach as low as 0.5 fM, which has improved by as much as 8 orders of magnitude as compared with the label-free SERS method24b and 6 orders of magnitude as compared with the previously reported SERS-based direct assay,27 and 3 orders of magnitude as compared with the EXPAR-based electrochemical biosensor.28 EXPAR-based SERS may discriminate target miRNA from the miRNAs with high sequence homology, and can be used to simultaneously detect multiple microRNAs in lung cancer cells, having great potential for further application in early clinical diagnosis. This work was supported by the National Natural Science Foundation of China (Grant No. 21325523 and 21205128), the Award for the Hundred Talent Program of the Chinese Academy of Sciences, the Fund for Shenzhen Engineering Laboratory of
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Single-molecule Detection and Instrument Development (Grant No. (2012) 433), the funds from State Ministry of Health of China (Grant No. 201002009) and Peking University Third Hospital (Grant No. 2013-BYSY-CAS-007).
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