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Cite this: Chem. Commun., 2014, 50, 7160 Received 19th March 2014, Accepted 14th April 2014
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A quantum dot-based microRNA nanosensor for point mutation assays† Ya-ping Zeng,‡a Guichi Zhu,‡a Xiao-yun Yang,‡b Jun Cao,‡c Zhi-liang Jingc and Chun-yang Zhang*a
DOI: 10.1039/c4cc02034k www.rsc.org/chemcomm
We have developed a quantum dot-based microRNA nanosensor for point mutation assays using primer generation-mediated rolling circle amplification. The proposed method exhibits high sensitivity with a detection limit of as low as 50.9 aM and a large dynamic range of 7 orders of magnitude from 0.1 fM to 1 nM. Importantly, this method can be further applied to analyze the point mutation of mir-196a2 in the lung tissues of non small-cell lung cancer patients.
Lung cancer is the leading cause of cancer-related mortality, accounting for one third of all deaths from cancer worldwide.1 Tobacco smoking, atmospheric contamination and long-term exposure to environments containing carcinogenic substances are three primary pathogeneses of lung cancers.2 Like most carcinomas, lung cancer is a conglomeration of diseases with different subtypes including small-cell lung cancer (SCLC, 20% of lung cancers) and non small-cell lung cancer (NSCLC, 80% of lung cancers).3 Recent research demonstrates that lung cancers frequently develop through sequential morphological steps by the accumulation of multiple genetic alterations, and that endogenous non-coding microRNAs (miRNAs) might function as potential tumor suppressors/oncogenes for lung cancer.4 miRNAs are a class of short RNA molecules with a length of 17–24 nucleotides, and they play important roles in numerous cellular processes, such as differentiation, cell growth, and apoptosis.5 The aberrant expression of miRNAs in the human genome is usually associated with a variety of cancers,6 and the point mutation of miRNAs might affect the cancer risk, the treatment efficacy, and even the survival of individual NSCLC patients.7 Therefore, the development of a reliable and sensitive a
Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China. E-mail: [email protected]; Fax: +86-755-86392299; Tel: +86-755-86392211 b Department of Rehabilitation Medicine, Affiliated Hospital of Guangdong Medical College, Zhanjiang 524001, China c Department of Pathology, Affiliated Hospital of Guangdong Medical College, Zhanjiang 524001, China † Electronic supplementary information (ESI) available: Details of experimental procedures and additional figures. See DOI: 10.1039/c4cc02034k ‡ These authors contributed equally.
7160 | Chem. Commun., 2014, 50, 7160--7162
method for a miRNA point mutation assay is highly desired for the early diagnosis of NSCLC. The quantitative real-time polymerase chain reaction (qRT-PCR) is usually used for miRNA point mutation assays due to its high sensitivity and practicality.8 However, qRT-PCR can involve several complex processes including reverse transcription, multiple primer design, and precise temperature control.8 In addition, nonspecific amplification might induce false positive results.9 Recently, some new methods, such as the locked nucleic acid-based assay,10 padlock probe-based exponential rolling circle amplification,11 and the size-coded ligation chain reaction,12 have been developed to improve the detection specificity, but the involvement of immobilization/separation steps,10 time-consuming reaction processes,11 and the complicated design of multiple DNA probes12 limit their practical applications. Consequently, the development of sensitive and simple methods for a miRNA point mutation assay still remains a great challenge. Quantum dots (QDs) are novel semiconductor nanocrystals with unique optical properties, including size-tunable photoluminescence spectra and relatively high quantum yield, and have been widely used as fluorescent markers in biological labelling, fluorescence imaging, and drug delivery.13 In particular, QDs hold great promise as fluorescence resonance energy transfer (FRET) donors in various biosensors to homogeneously detect nucleic acids, proteins, and small molecules.14 The combination of QDs with single-molecule detection enables the development of QD-based biosensors with distinct advantages including high signal-to-noise ratios, low sample consumption, and high sensitivity.15 The QD-based biosensors can detect the target biomolecules at the single-particle level with a detection limit on the attomolar scale.16 Here, we develop a QD-based miRNA nanosensor for a point mutation assay using primer generation-mediated rolling circle amplification (PG-RCA), and further apply it to analyze the point mutation of mir-196a2 in the lung tissues of NSCLC patients. Recent research demonstrates that the miRNA point mutation in mir-196a2 (T - C) is associated with the individual survival of NSCLC patients, and the increased expression of mir-196a2C might lead to the shortened survival of NSCLC patients through
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Scheme 1 Schematic illustration of a QD-based miRNA nanosensor for a point mutation assay.
altering the binding activity of target mRNA.7a,17,18 The principle of the miRNA point mutation assay is illustrated in Scheme 1. In the presence of mir-196a2T (Scheme 1A), the 30 - and 50 - terminal of the mir-196a2T-specific linear padlock probe (see ESI,† Table S1) can be ligated to form a circular template, and mir-196a2T can function as a primer to initiate the RCA reaction in the presence of Vent (exo-) polymerase, producing a long repeat sequence. This long repeat sequence can be nicked by Nb.BsmI, generating abundant new primers to initiate a new RCA reaction, consequently leading to an exponential amplification. The amplified DNA products can hybridize with the biotin/Cy5-labeled capture probes to form a double-stranded DNA (dsDNA) with a recognition site of the Nt.BstNBI nicking enzyme. After the nicking reaction, the capture probe is cleaved, leading to the separation of Cy5 from biotin and the release of the amplified product. The released amplified product can repeatedly hybridize with new biotin/Cy5-labeled capture probes to initiate the next cycle of cleavage, inducing the cleavage of the abundant biotin/Cy5-labeled capture probes. The higher the mir-196a2T concentration, the less the Cy5 molecules are absorbed onto the surface of the streptavidincoated QD. Consequently this reduces the FRET efficiency between the QD donor and Cy5 acceptors, and results in fewer Cy5 counts being detected. While in the presence of mir-196a2C (Scheme 1B), the mir-196a2T-specific linear padlock probe cannot be circularized, and no amplification reaction is triggered. Thus, the biotin/Cy5-labeled capture DNA probe will keep its single strand, and cannot be cleaved by the Nt.BstNBI nicking enzyme. With the addition of the streptavidin-coated QDs, the biotin/Cy5-labeled capture probes will be assembled on the surface of QDs to form the QD–capture probe–Cy5 complex through a specific streptavidin-biotin interaction, resulting in the occurrence of FRET between the QD donor and Cy5 acceptors and consequently the detection of Cy5 counts. Through measuring the reduction of Cy5 counts induced by mir-196a2T, we can accurately quantify the mir-196a2T concentration. In addition, a similar approach can be used to detect mir-196a2C using the mir196a2C-specific linear padlock probes (see ESI,† Table S1). Fig. 1 shows the fluorescence images of a QD-based miRNA nanosensor using the mir-196a2T-specific linear padlock probes in
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Fig. 1 Fluorescence images of the QD-based miRNA nanosensor using the mir-196a2T-specific linear padlock probes in the presence of mir-196a2T (A, B and C) and mir-196a2C (D, E and F), respectively. The fluorescence images of the QDs (A and D) are shown in green, and the fluorescence images of Cy5 (B and E) are shown in red. The fluorescence images with blended colours in yellow and orange (F) indicate the co-localization of the QDs and Cy5. The scale bar is 2 mm.
the presence of mir-196a2T (Fig. 1A–C) and mir-196a2C (Fig. 1D–F), respectively. In the presence of mir-196a2T, only the QD fluorescence signal (Fig. 1A) is observed, without any Cy5 fluorescence signal (Fig. 1B), suggesting that no FRET occurs between the QD donor and the Cy5 acceptor. This can be explained by the fact that the dsDNA formed by the binding of the capture probe with the amplified product can be recognized and cleaved by the Nt.BstNBI nicking enzyme, resulting in the separation of the Cy5 acceptor from the QD donor. While in the presence of mir-196a2C, both QD (Fig. 1D) and Cy5 (Fig. 1E) fluorescence signals are observed due to efficient FRET between QD donor and Cy5 acceptors in the QD–capture probe–Cy5 complex. It is worth noting that the QD fluorescence signals in the presence of mir-196a2C (Fig. 1D) are much weaker than those in the presence of mir-196a2T (Fig. 1A). This can be explained by the quenching of the QD fluorescence as a result of energy transfer from the QD donor to the Cy5 acceptor due to the formation of the QD–capture probe–Cy5 complex in the presence of mir-196a2C (Scheme 1B). To evaluate the detection sensitivity of the QD-based miRNA nanosensor for the point mutation assay, we further measured mir196a2T at various concentrations using the mir-196a2T-specific linear padlock probes under optimal conditions (see ESI,† Fig. S1 and S2). As shown in Fig. S3A (see ESI†), the reduction of the Cy5 counts increases with the increasing concentration of mir-196a2T. Using a logarithmic scale, the reduction of Cy5 counts exhibits a linear correlation with the concentration of mir-196a2T over a large dynamic range of 7 orders of magnitude from 0.1 fM to 1 nM (see ESI,† Fig. S3B). The regression equation is Nreduction = 1018.9 + 62.1 log10 c with a correlation coefficient of 0.996, where Nreduction is the reduction of Cy5 counts and c is the concentration of mir-196a2T, respectively. The detection limit is calculated to be 50.9 aM by evaluating the average signal of the blank plus three
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times the standard deviation. Notably, the sensitivity of the proposed method improved by 8 orders of magnitude as compared with the hairpin RNAzyme cleavage-based method (5 nM),19 and by more than 4 orders of magnitude as compared with the size-coded ligation-mediated PCR method (1 pM).20 The improved sensitivity of the proposed method can be attributed to the high amplification efficiency of the PG-RCA reaction, the amplified signal induced by the repeated cleavage of the Nt.BstNBI nicking enzyme, and the high signal-to-noise ratio of the single molecule detection method. Moreover, the proposed method can even distinguish variant frequencies as low as 0.001% in the artificial mixtures of mir196a2T and mir-196a2C (see ESI,† Fig. S4). Recent research demonstrates that the aberrant expressions of miRNAs might function as valuable biomarkers for human cancer prognosis,21 and the point mutation of mir-196a2 (T - C) is associated with the individual survival of NSCLC patients.7a,17,18 To investigate the feasibility of the QD-based miRNA nanosensor in clinical diagnosis, we measured the expression of mir-196a2T and mir-196a2C in total RNA samples obtained from 24 NSCLC patients using the specific linear padlock probes. The total RNA was extracted from the lung tissues using the miRNeasy FFPE kit (Qiagen) according to the manufacturer’s procedures. With the mir-196a2Tspecific linear padlock probe as the template, the reduction of Cy5 counts indicates the expression level of mir-196a2T. The greater the reduction of Cy5 counts, the higher the expression level of mir196a2T (Fig. 2A). With the mir-196a2C-specific linear padlock probe as the template, the reduction of Cy5 counts represents the expression level of mir-196a2C (Fig. 2B). As shown in Fig. 2, the expression of mir-196a2T in patients 1–7 is significantly lower than in patients 8–24 (Fig. 2A, unpaired t test, P o 0.001), but the expression of mir196a2C in patients 1–7 is significantly higher than in patients 8–24 (Fig. 2B, unpaired t test, P o 0.001), suggesting the selective expression of mir-196a2C and mir-196a2T in NSCLC patients.7b Based on previous research,7a,17,18 patients 1–7 with an increased expression of mir-196a2C might suffer from a shorter survival time than patients 8–24. It is worth noting that the conclusion about the individual survival time should be verified by the tractable clinical data which requires monitoring for several years. In summary, we have developed a QD-based miRNA nanosensor which may be used as a point mutation assay using primer generation-mediated rolling circle amplification. Owing to the excellent specificity of the line padlock probes and T4 RNA ligase 2,12,22 the proposed method can discriminate the point mutation between
Fig. 2 Expression of mir-196a2T (A) and mir-196a2C (B) in 24 NSCLC patients analyzed by the QD-based miRNA nanosensor. Error bars show the standard deviation of three experiments.
7162 | Chem. Commun., 2014, 50, 7160--7162
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mir-196a2T and mir-196a2C. With the integration of the PG-RCA reaction and nicking enzyme-driven recycling amplification, this QDbased miRNA nanosensor exhibits high sensitivity with a detection limit of as low as 50.9 aM and a large dynamic range of 7 orders of magnitude from 0.1 fM to 1 nM. Importantly, this QD-based miRNA nanosensor can be used to analyze the point mutation of mir-196a2 in the lung tissues of NSCLC patients, holding great potential for further application in biomedical research and clinical diagnosis. This work was supported by the National Natural Science Foundation of China (Grant No. 21325523), the Award for the Hundred Talent Program of the Chinese Academy of Sciences, and the Fund for Shenzhen Engineering Laboratory of Single-Molecule Detection and Instrument Development (Grant No. (2012)433).
Notes and references 1 S. V. Sharma, D. W. Bell, J. Settleman and D. A. Haber, Nat. Rev. Cancer, 2007, 7, 169. 2 (a) N. Yanaihara, N. Caplen, E. Bowman, M. Seike, K. Kumamoto, M. Yi, R. M. Stephens, A. Okamoto, J. Yokota, T. Tanaka, G. A. Calin, C. G. Liu, C. M. Croce and C. C. Harris, Cancer Cell, 2006, 9, 189; (b) C. A. Pope, R. T. Burnett, M. J. Thun, E. E. Calle, D. Krewski, I. Kazuhiko and G. D. Thurston, JAMA, J. Am. Med. Assoc., 2002, 287, 1132. 3 A. Jemal, F. Bray, M. M. Center, J. Ferlay, E. Ward and D. Forman, Ca-Cancer J. Clin., 2011, 61, 69. 4 M. Boeri, C. Verri, D. Conte, L. Roz, P. Modena, F. Facchinetti, E. Calabro, C. M. Croce, U. Pastorino and G. Sozzi, Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 3713. 5 A. M. Cheng, M. W. Byrom, J. Shelton and L. P. Ford, Nucleic Acids Res., 2005, 33, 1290. 6 G. A. Calin and C. M. Croce, Nat. Rev. Cancer, 2006, 6, 857. 7 (a) Z. Hu, J. Chen, T. Tian, X. Zhou, H. Gu, L. Xu, Y. Zeng, R. Miao, G. Jin, H. Ma, Y. Chen and H. Shen, J. Clin. Invest., 2008, 118, 2600; (b) B. M. Ryan, A. I. Robles and C. C. Harris, Nat. Rev. Cancer, 2010, 10, 389. 8 L. J. Chin, E. Ratner, S. Leng, R. Zhai, S. Nallur, I. Babar, R. U. Muller, E. Straka, L. Su, E. A. Burki, R. E. Crowell, R. Patel, T. Kulkarni, R. Homer, D. Zelterman, K. K. Kidd, Y. Zhu, D. C. Christiani, S. A. Belinsky, F. J. Slack and J. B. Weidhaas, Cancer Res., 2008, 68, 8535. 9 Q. Su, D. Xing and X. Zhou, Biosens. Bioelectron., 2010, 25, 1615. 10 (a) J. M. Lee and Y. Jung, Angew. Chem., Int. Ed., 2011, 50, 12487; (b) S. Karmakar and P. J. Hrdlicka, Chem. Sci., 2013, 4, 3447. 11 H. Liu, L. Li, L. Duan, X. Wang, Y. Xie, L. Tong, Q. Wang and B. Tang, Anal. Chem., 2013, 85, 7941. 12 P. Zhang, J. Zhang, C. Wang, C. Liu, H. Wang and Z. Li, Anal. Chem., 2014, 86, 1076. 13 (a) I. L. Medintz, H. T. Uyeda, E. Goldman and H. Mattoussi, Nat. Mater., 2005, 4, 435; (b) R. C. Somers, M. G. Bawendi and D. G. Nocera, Chem. Soc. Rev., 2007, 36, 579; (c) Q. Wang, Y. Liu, Y. Ke and H. Yan, Angew. Chem., Int.Ed., 2008, 47, 316. 14 (a) I. L. Medintz and H. Mattoussi, Phys. Chem. Chem. Phys., 2009, 11, 17; (b) K. Boeneman, B. C. Mei, A. M. Dennis, G. Bao, J. R. Deschamps, H. Mattoussi and I. L. Medintz, J. Am. Chem. Soc., 2009, 131, 3828. 15 C. Y. Zhang, H. C. Yeh, M. T. Kuroki and T. H. Wang, Nat. Mater., 2005, 4, 826. 16 J. Zhou, Q. X. Wang and C. Y. Zhang, J. Am. Chem. Soc., 2013, 135, 2056. 17 T. Tian, Y. Shu, J. Chen, Z. Hu, L. Xu, G. Jin, J. Liang, P. Liu, X. Zhou, R. Miao, H. Ma and Y. Chen, Cancer Epidemiol., Biomarkers Prev., 2009, 18, 1183. 18 M. Bloomston, W. L. Frankel, F. Petrocca, S. Volinia, H. Alder, J. P. Hagan, C. G. Liu, D. Bhatt, C. Taccioli and C. M. Croce, JAMA, J. Am. Med. Assoc., 2007, 297, 1901. 19 J. S. Hartig, I. Grune, S. H. Najafi-Shoushtari and M. Famulok, J. Am. Chem. Soc., 2004, 126, 722. 20 E. Arefian, J. Kiani, M. Soleimani, S. A. M. Shariati, S. H. AghaeeBakhtiari, A. Atashi, Y. Gheisari, N. Ahmadbeigi, A. M. BanaeiMoghaddam, M. Naderi, N. Namvaras, L. Good and O. R. Faridani, Nucleic Acids Res., 2011, 39, e80. 21 A. Esquela-Kerscher and F. J. Slack, Nat. Rev. Cancer, 2006, 6, 259. 22 Y. Cheng, X. Zhang, Z. Li, X. Jiao, Y. Wang and Y. Zhang, Angew. Chem., Int. Ed., 2009, 48, 3268.
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Cite this: Chem. Commun., 2014, 50, 7160 Received 19th March 2014, Accepted 14th April 2014
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A quantum dot-based microRNA nanosensor for point mutation assays† Ya-ping Zeng,‡a Guichi Zhu,‡a Xiao-yun Yang,‡b Jun Cao,‡c Zhi-liang Jingc and Chun-yang Zhang*a
DOI: 10.1039/c4cc02034k www.rsc.org/chemcomm
We have developed a quantum dot-based microRNA nanosensor for point mutation assays using primer generation-mediated rolling circle amplification. The proposed method exhibits high sensitivity with a detection limit of as low as 50.9 aM and a large dynamic range of 7 orders of magnitude from 0.1 fM to 1 nM. Importantly, this method can be further applied to analyze the point mutation of mir-196a2 in the lung tissues of non small-cell lung cancer patients.
Lung cancer is the leading cause of cancer-related mortality, accounting for one third of all deaths from cancer worldwide.1 Tobacco smoking, atmospheric contamination and long-term exposure to environments containing carcinogenic substances are three primary pathogeneses of lung cancers.2 Like most carcinomas, lung cancer is a conglomeration of diseases with different subtypes including small-cell lung cancer (SCLC, 20% of lung cancers) and non small-cell lung cancer (NSCLC, 80% of lung cancers).3 Recent research demonstrates that lung cancers frequently develop through sequential morphological steps by the accumulation of multiple genetic alterations, and that endogenous non-coding microRNAs (miRNAs) might function as potential tumor suppressors/oncogenes for lung cancer.4 miRNAs are a class of short RNA molecules with a length of 17–24 nucleotides, and they play important roles in numerous cellular processes, such as differentiation, cell growth, and apoptosis.5 The aberrant expression of miRNAs in the human genome is usually associated with a variety of cancers,6 and the point mutation of miRNAs might affect the cancer risk, the treatment efficacy, and even the survival of individual NSCLC patients.7 Therefore, the development of a reliable and sensitive a
Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China. E-mail: [email protected]; Fax: +86-755-86392299; Tel: +86-755-86392211 b Department of Rehabilitation Medicine, Affiliated Hospital of Guangdong Medical College, Zhanjiang 524001, China c Department of Pathology, Affiliated Hospital of Guangdong Medical College, Zhanjiang 524001, China † Electronic supplementary information (ESI) available: Details of experimental procedures and additional figures. See DOI: 10.1039/c4cc02034k ‡ These authors contributed equally.
7160 | Chem. Commun., 2014, 50, 7160--7162
method for a miRNA point mutation assay is highly desired for the early diagnosis of NSCLC. The quantitative real-time polymerase chain reaction (qRT-PCR) is usually used for miRNA point mutation assays due to its high sensitivity and practicality.8 However, qRT-PCR can involve several complex processes including reverse transcription, multiple primer design, and precise temperature control.8 In addition, nonspecific amplification might induce false positive results.9 Recently, some new methods, such as the locked nucleic acid-based assay,10 padlock probe-based exponential rolling circle amplification,11 and the size-coded ligation chain reaction,12 have been developed to improve the detection specificity, but the involvement of immobilization/separation steps,10 time-consuming reaction processes,11 and the complicated design of multiple DNA probes12 limit their practical applications. Consequently, the development of sensitive and simple methods for a miRNA point mutation assay still remains a great challenge. Quantum dots (QDs) are novel semiconductor nanocrystals with unique optical properties, including size-tunable photoluminescence spectra and relatively high quantum yield, and have been widely used as fluorescent markers in biological labelling, fluorescence imaging, and drug delivery.13 In particular, QDs hold great promise as fluorescence resonance energy transfer (FRET) donors in various biosensors to homogeneously detect nucleic acids, proteins, and small molecules.14 The combination of QDs with single-molecule detection enables the development of QD-based biosensors with distinct advantages including high signal-to-noise ratios, low sample consumption, and high sensitivity.15 The QD-based biosensors can detect the target biomolecules at the single-particle level with a detection limit on the attomolar scale.16 Here, we develop a QD-based miRNA nanosensor for a point mutation assay using primer generation-mediated rolling circle amplification (PG-RCA), and further apply it to analyze the point mutation of mir-196a2 in the lung tissues of NSCLC patients. Recent research demonstrates that the miRNA point mutation in mir-196a2 (T - C) is associated with the individual survival of NSCLC patients, and the increased expression of mir-196a2C might lead to the shortened survival of NSCLC patients through
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Scheme 1 Schematic illustration of a QD-based miRNA nanosensor for a point mutation assay.
altering the binding activity of target mRNA.7a,17,18 The principle of the miRNA point mutation assay is illustrated in Scheme 1. In the presence of mir-196a2T (Scheme 1A), the 30 - and 50 - terminal of the mir-196a2T-specific linear padlock probe (see ESI,† Table S1) can be ligated to form a circular template, and mir-196a2T can function as a primer to initiate the RCA reaction in the presence of Vent (exo-) polymerase, producing a long repeat sequence. This long repeat sequence can be nicked by Nb.BsmI, generating abundant new primers to initiate a new RCA reaction, consequently leading to an exponential amplification. The amplified DNA products can hybridize with the biotin/Cy5-labeled capture probes to form a double-stranded DNA (dsDNA) with a recognition site of the Nt.BstNBI nicking enzyme. After the nicking reaction, the capture probe is cleaved, leading to the separation of Cy5 from biotin and the release of the amplified product. The released amplified product can repeatedly hybridize with new biotin/Cy5-labeled capture probes to initiate the next cycle of cleavage, inducing the cleavage of the abundant biotin/Cy5-labeled capture probes. The higher the mir-196a2T concentration, the less the Cy5 molecules are absorbed onto the surface of the streptavidincoated QD. Consequently this reduces the FRET efficiency between the QD donor and Cy5 acceptors, and results in fewer Cy5 counts being detected. While in the presence of mir-196a2C (Scheme 1B), the mir-196a2T-specific linear padlock probe cannot be circularized, and no amplification reaction is triggered. Thus, the biotin/Cy5-labeled capture DNA probe will keep its single strand, and cannot be cleaved by the Nt.BstNBI nicking enzyme. With the addition of the streptavidin-coated QDs, the biotin/Cy5-labeled capture probes will be assembled on the surface of QDs to form the QD–capture probe–Cy5 complex through a specific streptavidin-biotin interaction, resulting in the occurrence of FRET between the QD donor and Cy5 acceptors and consequently the detection of Cy5 counts. Through measuring the reduction of Cy5 counts induced by mir-196a2T, we can accurately quantify the mir-196a2T concentration. In addition, a similar approach can be used to detect mir-196a2C using the mir196a2C-specific linear padlock probes (see ESI,† Table S1). Fig. 1 shows the fluorescence images of a QD-based miRNA nanosensor using the mir-196a2T-specific linear padlock probes in
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Fig. 1 Fluorescence images of the QD-based miRNA nanosensor using the mir-196a2T-specific linear padlock probes in the presence of mir-196a2T (A, B and C) and mir-196a2C (D, E and F), respectively. The fluorescence images of the QDs (A and D) are shown in green, and the fluorescence images of Cy5 (B and E) are shown in red. The fluorescence images with blended colours in yellow and orange (F) indicate the co-localization of the QDs and Cy5. The scale bar is 2 mm.
the presence of mir-196a2T (Fig. 1A–C) and mir-196a2C (Fig. 1D–F), respectively. In the presence of mir-196a2T, only the QD fluorescence signal (Fig. 1A) is observed, without any Cy5 fluorescence signal (Fig. 1B), suggesting that no FRET occurs between the QD donor and the Cy5 acceptor. This can be explained by the fact that the dsDNA formed by the binding of the capture probe with the amplified product can be recognized and cleaved by the Nt.BstNBI nicking enzyme, resulting in the separation of the Cy5 acceptor from the QD donor. While in the presence of mir-196a2C, both QD (Fig. 1D) and Cy5 (Fig. 1E) fluorescence signals are observed due to efficient FRET between QD donor and Cy5 acceptors in the QD–capture probe–Cy5 complex. It is worth noting that the QD fluorescence signals in the presence of mir-196a2C (Fig. 1D) are much weaker than those in the presence of mir-196a2T (Fig. 1A). This can be explained by the quenching of the QD fluorescence as a result of energy transfer from the QD donor to the Cy5 acceptor due to the formation of the QD–capture probe–Cy5 complex in the presence of mir-196a2C (Scheme 1B). To evaluate the detection sensitivity of the QD-based miRNA nanosensor for the point mutation assay, we further measured mir196a2T at various concentrations using the mir-196a2T-specific linear padlock probes under optimal conditions (see ESI,† Fig. S1 and S2). As shown in Fig. S3A (see ESI†), the reduction of the Cy5 counts increases with the increasing concentration of mir-196a2T. Using a logarithmic scale, the reduction of Cy5 counts exhibits a linear correlation with the concentration of mir-196a2T over a large dynamic range of 7 orders of magnitude from 0.1 fM to 1 nM (see ESI,† Fig. S3B). The regression equation is Nreduction = 1018.9 + 62.1 log10 c with a correlation coefficient of 0.996, where Nreduction is the reduction of Cy5 counts and c is the concentration of mir-196a2T, respectively. The detection limit is calculated to be 50.9 aM by evaluating the average signal of the blank plus three
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times the standard deviation. Notably, the sensitivity of the proposed method improved by 8 orders of magnitude as compared with the hairpin RNAzyme cleavage-based method (5 nM),19 and by more than 4 orders of magnitude as compared with the size-coded ligation-mediated PCR method (1 pM).20 The improved sensitivity of the proposed method can be attributed to the high amplification efficiency of the PG-RCA reaction, the amplified signal induced by the repeated cleavage of the Nt.BstNBI nicking enzyme, and the high signal-to-noise ratio of the single molecule detection method. Moreover, the proposed method can even distinguish variant frequencies as low as 0.001% in the artificial mixtures of mir196a2T and mir-196a2C (see ESI,† Fig. S4). Recent research demonstrates that the aberrant expressions of miRNAs might function as valuable biomarkers for human cancer prognosis,21 and the point mutation of mir-196a2 (T - C) is associated with the individual survival of NSCLC patients.7a,17,18 To investigate the feasibility of the QD-based miRNA nanosensor in clinical diagnosis, we measured the expression of mir-196a2T and mir-196a2C in total RNA samples obtained from 24 NSCLC patients using the specific linear padlock probes. The total RNA was extracted from the lung tissues using the miRNeasy FFPE kit (Qiagen) according to the manufacturer’s procedures. With the mir-196a2Tspecific linear padlock probe as the template, the reduction of Cy5 counts indicates the expression level of mir-196a2T. The greater the reduction of Cy5 counts, the higher the expression level of mir196a2T (Fig. 2A). With the mir-196a2C-specific linear padlock probe as the template, the reduction of Cy5 counts represents the expression level of mir-196a2C (Fig. 2B). As shown in Fig. 2, the expression of mir-196a2T in patients 1–7 is significantly lower than in patients 8–24 (Fig. 2A, unpaired t test, P o 0.001), but the expression of mir196a2C in patients 1–7 is significantly higher than in patients 8–24 (Fig. 2B, unpaired t test, P o 0.001), suggesting the selective expression of mir-196a2C and mir-196a2T in NSCLC patients.7b Based on previous research,7a,17,18 patients 1–7 with an increased expression of mir-196a2C might suffer from a shorter survival time than patients 8–24. It is worth noting that the conclusion about the individual survival time should be verified by the tractable clinical data which requires monitoring for several years. In summary, we have developed a QD-based miRNA nanosensor which may be used as a point mutation assay using primer generation-mediated rolling circle amplification. Owing to the excellent specificity of the line padlock probes and T4 RNA ligase 2,12,22 the proposed method can discriminate the point mutation between
Fig. 2 Expression of mir-196a2T (A) and mir-196a2C (B) in 24 NSCLC patients analyzed by the QD-based miRNA nanosensor. Error bars show the standard deviation of three experiments.
7162 | Chem. Commun., 2014, 50, 7160--7162
Communication
mir-196a2T and mir-196a2C. With the integration of the PG-RCA reaction and nicking enzyme-driven recycling amplification, this QDbased miRNA nanosensor exhibits high sensitivity with a detection limit of as low as 50.9 aM and a large dynamic range of 7 orders of magnitude from 0.1 fM to 1 nM. Importantly, this QD-based miRNA nanosensor can be used to analyze the point mutation of mir-196a2 in the lung tissues of NSCLC patients, holding great potential for further application in biomedical research and clinical diagnosis. This work was supported by the National Natural Science Foundation of China (Grant No. 21325523), the Award for the Hundred Talent Program of the Chinese Academy of Sciences, and the Fund for Shenzhen Engineering Laboratory of Single-Molecule Detection and Instrument Development (Grant No. (2012)433).
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