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Cite this: DOI: 10.1039/c5cc01317h Received 12th February 2015, Accepted 17th March 2015
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Chemiluminescence detection of DNA/microRNA based on cation-exchange of CuS nanoparticles and rolling circle amplification† Xiaoru Zhang,*a Hongxia Liu,a Ruijuan Li,a Ningbo Zhang,b Ying Xionga and Shuyan Niua
DOI: 10.1039/c5cc01317h www.rsc.org/chemcomm
Cation-exchange-based chemiluminescence amplification (CXCLAmp) was developed in this study. After amplification by rolling circle amplification (RCA), the proposed RCA–CXCLAmp strategy was used to detect miRNA sensitively.
As a highly sensitive and convenient tool, chemiluminescence (CL) has been used in diverse fields, such as disease diagnosis, drug screening, forensic activation analysis and environmental monitoring.1 The oxidation of luminol with H2O2 is a popular CL reaction. To enhance the CL intensity of such a reaction, some catalysts have been suggested, including enzymes,2 DNAzymes,3 transition metal ions4 and nanoparticles.5 Among them, nanoparticles used as biological tags, for example labeled on DNA hybrids or immune complexes, enable new possibilities for biological analysis due to their unique optical and chemical properties. Labelled nanoparticles can be dissolved under acidic conditions producing a large quantity of the corresponding metal ions.5a,6 However, the strong acid HNO3 may destroy biological specimens, as a result, it is unfavourable for recycling the biosensor. On the other hand, the oxidation of luminol with H2O2 is always carried out in a strongly basic medium, which is in conflict with such strong acidic conditions. Recently, Son et al. reported a cation exchange reaction of ionic nanocrystals (NCs).7 This reaction is reversible and can complete in a short period of time at room temperature. Later, Zhong’s group published a series of reports on a cation-exchangebased fluorescence amplification method (CXFluoAmp).8 By exchanging non-fluorescent CdSe or ZnS NC clusters with Ag+, Ag2Se NCs, together with Cd2+ or Zn2+ cations, were formed.
The generated cations can coordinate to metal-responsive fluorophores and activate fluorogenic dyes. Each ionic NC, with a diameter of a few nanometres, can encapsulate thousands of corresponding ions; for example, each ZnSe NC, with a calculated diameter of 5.38 nm, contains 2051 Zn atoms.9 Therefore, a large signal enhancement may be realised through this fast and gentle process. However, to date, the application of this highly efficient reaction has been restricted to fluorescence detection. Inspired by this simple and convenient reaction, here, we attempt to apply cation exchange reaction to a CL sensor. b-Mercaptopropionic acid modified nano-CuS was selected as a tag due to the highly efficient catalytic ability of cupric ions during the CL assay and good biocompatibility of CuS NPs.10 Because Ksp of Ag2S is 6.69 10 50 (a) or 1.09 10 49 (b), while Ksp of CuS is 1.27 10 36, it is easy for Ag+ ions to exchange with Cu2+ ions. Here, for the first time, the cation-exchange reaction between CuS NPs and AgNO3 was presented. To test the feasibility of the proposed CXCLAmp strategy, a simple sandwich model was designed as shown in Fig. 1. The capture DNA was assembled onto the surface of magnetic beads (MBs). After hybridising with the target DNA and the CuS NP-labelled reporter DNA, a sandwich structure was formed with a CuS NP tag. Then, a solution of AgNO3 was added to the conjugates, and the supernatant was subjected to CL measurement. As shown in Fig. 2, in the absence of the target DNA, CuS NPs could not be introduced, as expected, and
a
Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, P. R. China. E-mail: [email protected] b Key Laboratory of Detection Technology of Shandong Province for Tumor Marker, College of Chemistry and Chemical Engineering, Linyi University, Linyi 276005, P. R. China † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c5cc01317h
This journal is © The Royal Society of Chemistry 2015
Fig. 1 Schematic illustration of the cation-exchange-based chemiluminescence assay and its use in detecting DNA through sandwich assembly.
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Fig. 2 CL kinetic response in the presence (a) and absence (b) of 4.0 10 12 M target DNA.
Fig. 3 The CL reaction time under different conditions. Cu2+ was produced by cation-exchange with AgNO3 or an oxidation reaction with nitric acid. The concentration of the target DNA was 1.0 10 12 M.
the CL intensity was negligible (curve b). However, the CL intensity increased substantially in the presence of 4.0 10 12 M target DNA (curve a), indicating that a large amount of Cu2+ ions was produced. To further verify the feasibility of the cation-exchange reaction, inductively coupled plasma-mass spectrometry (ICP-MS) was used to detect the produced Cu2+ directly (see Table 1). At the same time, the influence of Ag+ ions on the CL reaction was investigated carefully. Fig. S4 (ESI†) showed that Ag+ showed almost no effect on the luminol–H2O2 system or the Cu2+–luminol–H2O2 system across a wide concentration range (from 4.0 10 4 M to 4.0 10 16 M) due to the relatively weak oxidation effect of AgNO3.1a So, we can conclude that the CXCLAmp strategy works well and can be used to determine the target content. The time taken for the completion of the cation-exchange reaction was compared with that of oxidation by HNO3 (0.16 M, 10 mL). The results in Fig. 3 show that both reactions could complete within a few minutes at room temperature. But the CL signal of oxidation by HNO3 was much lower when using the same concentration of target DNA. This effect may be explained because the strong acid HNO3 is unfavourable for the reaction between luminol and H2O2, which is usually carried out in a strongly basic medium. Therefore, the detection limit of this method (1.0 10 14 M, see the ESI†) is approximately 50 times lower than that of a similar sandwich CL detection reported by Zhang et al.,6a who applied strong acid and even amplified by the anodic stripping voltammetry technique. The regeneration ability is an important property for a biosensor. Unlike oxidation by nitric acid, which could destroy the structure of DNA completely, the CXCLAmp strategy can be used for the continuous detection of the target DNA after 3 or 4 regeneration cycles (see Fig. S5, ESI†) due to the highly compatible reaction conditions. Moreover, the proposed CXCLAmp strategy can also be used in the regeneration of biosensors that distinguish between antigens and antibodies as well as between carbohydrates and lectins.
On the base of feasibility study for the CXCLAmp strategy, we further decided to detect miRNA using this method, coupled with rolling circle amplification for signal amplification. The RCA reaction, which utilises DNA polymerases to generate a long single DNA strand with repeated sequences,11 was employed for introducing a large number of CuS NP tags. As shown in Fig. 4A, this RCA-amplified CXCLAmp strategy began with immobilising the capture DNA, comprising a miRNA recognition segment and a universal adapter segment. Without the target miRNA, the ligated adapter could not hybridise with the capture DNA on the MB because the melting temperature of such a double-stranded structure was below the reaction temperature (45 1C). However, in the presence of the target miRNA, the ligated adapter could link to miRNA using T4 ligase. Then, the ligated adapter could be extended through RCA when reacted with padlock DNA, T4 ligase and phi29 polymerase. The obtained long sequence, with many repeated copies, facilitates incorporating a large number of CuS NPs after hybridising with the reporter DNA–CuS NP complex. When adding AgNO3, Cu2+ was released through a cation-exchange reaction, which could catalyse a CL reaction, as described above. Thus, such a RCA– CXCLAmp strategy created a cascade of signal enhancements for the sensitive detection of miRNA. To prove the amplification effect of the RCA reaction, the CL performances under different conditions were compared, as shown in Fig. 4B. When the simple sandwich CXCLAmp strategy was applied (shown in Fig. 1), compared with the background of RCA–CXCLAmp (curve a), an obvious enhancement of the CL intensity in the presence of 1.0 10 12 M DNA could be observed (curve b). However, using the RCA–CXCLAmp strategy, a much greater enhancement of the CL intensity was observed, even in the presence of 1.0 10 14 M miRNA (curve c). The experimental results indicated that the RCA reaction could substantially amplify the CL signal for CXCLAmp detection. From the ICP-MS measurement shown in Table 1, we could see that the content of cupric ions was related to the concentration
Table 1
The total amount of Cu2+ released to solution after the cation–exchange reaction measured by ICP-MS
Concentration of DNA/RNA (M) 2+
Cu concentration for CXCLAmp (M) Cu2+ concentration for RCA–CXCLAmp (M)
Chem. Commun.
0 o0.000 o0.000
10
12
M
2.68 10 9.60 10
10 7 5
10
M
2.18 10 3.23 10
5 4
This journal is © The Royal Society of Chemistry 2015
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Fig. 6 Specificity of miRNA detection using the RCA–CXCLAmp method. The concentration of let-7a–g and let-7f each is 5 10 12 M.
Fig. 4 (A) Schematic illustration of the RCA-amplified CXCLAmp strategy and its use in the detection of miRNA; (B) the amplification effect of the RCA reaction. (a) Blank; (b) CXCLAmp strategy: the concentration of target DNA was 1.0 10 12 M; (c) RCA–CXCLAmp strategy: the concentration of target miRNA was 1.0 10 14 M.
of target DNA or miRNA. When more target DNAs or miRNAs were involved, a higher concentration of cupric ions was produced, which led to a higher CL signal. The amplification effect of the RCA reaction can also be demonstrated by comparing the content of cupric ions at the same concentrations of target DNA or miRNA. The detection capability of our RCA–CXCLAmp assay was determined using various concentrations of let-7a miRNA under optimum conditions. As shown in Fig. S11A and 11B (ESI†), the CL intensities of luminol–H2O2–Cu2+ increased with increasing let-7a concentration. The CL value exhibited a good linear relationship with the logarithm (lg) of the let-7a concentration
Fig. 5 The relative CL intensity in a log–linear correlation with the amount of let-7a in the range from 6.0 10 16 M to 5.0 10 11 M.
This journal is © The Royal Society of Chemistry 2015
over a wide dynamic range, from 6.0 10 16 M to 5.0 10 11 M (see Fig. 5). The correlation equation was I = 1.02 107 + 6.71 105 lgC (I represents the CL intensity; C is the concentration of the let-7a miRNA, n = 9, R2 = 0.9995). A detection limit of 0.17 fM (1.7 zmol) was estimated using 3s. The sensitivity of this strategy for miRNA detection could be comparable to those of most of the methods listed in Table S3 (ESI†). Moreover, after the amplification by RCA, the sensitivity of the RCA–CXCLAmp method increased approximately 100-fold compared with a simple CXCLAmp assay (see ESI†). Such a high sensitivity and wide linear range for the quantitative detection of let-7a miRNA could be attributed to the excellent performance of the CXCLAmp assay as well as the highly efficient RCA reaction. To assess the specificity of the strategy, let-7miRNA family members, which share the same lengths but differ by only 1–4 nucleotides from each other were chosen as controls. As shown in Fig. 6, all of the CL responses could be distinguished from that of the blank. The CL signal of let-7a was substantially higher than the others at the same concentration. Among these competitors, the sequences of let-7c, let-7e and let-7f have only one base mutation, indicating the excellent specificity of the developed miRNA assay. The analysis of let-7a in the 293T cell was also implemented to verify the accuracy and reliability of the method for real-world samples (see ESI†). In summary, a novel cation-exchange based chemiluminescence amplification assay was developed. Compared with the conventional CL methods, the proposed assay showed significant advantages in its low cost, nontoxic properties, ease of preparation and rapid response. The detection limit for DNA was 1.0 10 14 M using a simple sandwich strategy without any amplification process, due to the high efficiency of the cation exchange reaction, the excellent catalytic action of cupric ions for the CL reaction, and the highly compatible reaction conditions throughout the detection. Then, the RCA reaction was introduced for further enhancing the detection sensitivity. Under the optimum conditions, the detection limit for miRNA was 1.7 10 16 M with excellent specificity and could be used to detect miRNA in real-world samples. In the future, when combined with a flow-injection CL system, the performance of this strategy can be improved, making it more suitable for point-of-care detection.
Chem. Commun.
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This work was supported by the National Natural Science Foundation of China (No. 21275003); State Key Laboratory of Microbial Technology (M2013-13).
Published on 17 March 2015. Downloaded by UNIVERSITY OF NEBRASKA on 07/04/2015 12:45:38.
Notes and references 1 (a) S. Shinsuke, N. Atsushi, A. Kenji and A. Yasuhiro, J. Am. Chem. Soc., 2005, 127, 5300; (b) H. Cui, J. Guo, N. Li and L. Liu, J. Phys. Chem. C, 2008, 112, 11319. 2 E. Marzocchi, S. Grilli, L. Della-Ciana, L. Prodi, M. Mirasoli and A. Roda, Anal. Biochem., 2008, 377, 189. 3 L. Gong, Z. Zhao, Y. Lv, S. Huan, T. Fu, X. Zhang, G. Shen and R. Yu, Chem. Commun., 2015, 51, 979. 4 C. A. Chang and H. H. Patterson, Anal. Chem., 1980, 52, 653.
Chem. Commun.
ChemComm 5 (a) Z. Li, C. Liu, Y. Fan, Y. Wang and X. Duan, Anal. Biochem., 2006, 359, 247; (b) Y. He, D. Liu, X. He and H. Cui, Chem. Commun., 2011, 47, 10692. 6 (a) C. Ding, H. Zhong and S. Zhang, Biosens. Bioelectron., 2008, 23, 1314; (b) S. Zhang, H. Zhong and C. Ding, Anal. Chem., 2008, 80, 7206. 7 D. H. Son, S. M. Hughes, Y. Yin and A. P. Alivisatos, Science, 2004, 306, 1009. 8 (a) J. Li, T. Zhang, J. Ge, Y. Yin and W. Zhong, Angew. Chem., Int. Ed., 2009, 48, 1588; (b) J. Yao, S. Schachermeyer, Y. Yin and W. Zhong, Anal. Chem., 2011, 83, 402; (c) J. Yao, X. Han, S. Zeng and W. Zhong, Anal. Chem., 2012, 84, 1645; (d) J. Li, S. Schachermeyer, Y. Wang, Y. Yin and W. Zhong, Anal. Chem., 2009, 81, 9723; (e) J. Yao, K. Flack, L. Ding and W. Zhong, Analyst, 2013, 138, 3121. ¨ur, F. M. Boldt, S. Cavaliere-Jaricot, J. Ziegler and T. Nann, 9 E. Kucu Anal. Chem., 2007, 79, 8987. 10 K. Dong, Z. Liu, Z. Li, J. Ren and X. Qu, Adv. Mater., 2013, 25, 4452. 11 W. Zhao, M. M. Ali, M. A. Brook and Y. Li, Angew. Chem., Int. Ed., 2008, 47, 6330.
This journal is © The Royal Society of Chemistry 2015
Published on 17 March 2015. Downloaded by UNIVERSITY OF NEBRASKA on 07/04/2015 12:45:38.
COMMUNICATION
Cite this: DOI: 10.1039/c5cc01317h Received 12th February 2015, Accepted 17th March 2015
View Article Online View Journal
Chemiluminescence detection of DNA/microRNA based on cation-exchange of CuS nanoparticles and rolling circle amplification† Xiaoru Zhang,*a Hongxia Liu,a Ruijuan Li,a Ningbo Zhang,b Ying Xionga and Shuyan Niua
DOI: 10.1039/c5cc01317h www.rsc.org/chemcomm
Cation-exchange-based chemiluminescence amplification (CXCLAmp) was developed in this study. After amplification by rolling circle amplification (RCA), the proposed RCA–CXCLAmp strategy was used to detect miRNA sensitively.
As a highly sensitive and convenient tool, chemiluminescence (CL) has been used in diverse fields, such as disease diagnosis, drug screening, forensic activation analysis and environmental monitoring.1 The oxidation of luminol with H2O2 is a popular CL reaction. To enhance the CL intensity of such a reaction, some catalysts have been suggested, including enzymes,2 DNAzymes,3 transition metal ions4 and nanoparticles.5 Among them, nanoparticles used as biological tags, for example labeled on DNA hybrids or immune complexes, enable new possibilities for biological analysis due to their unique optical and chemical properties. Labelled nanoparticles can be dissolved under acidic conditions producing a large quantity of the corresponding metal ions.5a,6 However, the strong acid HNO3 may destroy biological specimens, as a result, it is unfavourable for recycling the biosensor. On the other hand, the oxidation of luminol with H2O2 is always carried out in a strongly basic medium, which is in conflict with such strong acidic conditions. Recently, Son et al. reported a cation exchange reaction of ionic nanocrystals (NCs).7 This reaction is reversible and can complete in a short period of time at room temperature. Later, Zhong’s group published a series of reports on a cation-exchangebased fluorescence amplification method (CXFluoAmp).8 By exchanging non-fluorescent CdSe or ZnS NC clusters with Ag+, Ag2Se NCs, together with Cd2+ or Zn2+ cations, were formed.
The generated cations can coordinate to metal-responsive fluorophores and activate fluorogenic dyes. Each ionic NC, with a diameter of a few nanometres, can encapsulate thousands of corresponding ions; for example, each ZnSe NC, with a calculated diameter of 5.38 nm, contains 2051 Zn atoms.9 Therefore, a large signal enhancement may be realised through this fast and gentle process. However, to date, the application of this highly efficient reaction has been restricted to fluorescence detection. Inspired by this simple and convenient reaction, here, we attempt to apply cation exchange reaction to a CL sensor. b-Mercaptopropionic acid modified nano-CuS was selected as a tag due to the highly efficient catalytic ability of cupric ions during the CL assay and good biocompatibility of CuS NPs.10 Because Ksp of Ag2S is 6.69 10 50 (a) or 1.09 10 49 (b), while Ksp of CuS is 1.27 10 36, it is easy for Ag+ ions to exchange with Cu2+ ions. Here, for the first time, the cation-exchange reaction between CuS NPs and AgNO3 was presented. To test the feasibility of the proposed CXCLAmp strategy, a simple sandwich model was designed as shown in Fig. 1. The capture DNA was assembled onto the surface of magnetic beads (MBs). After hybridising with the target DNA and the CuS NP-labelled reporter DNA, a sandwich structure was formed with a CuS NP tag. Then, a solution of AgNO3 was added to the conjugates, and the supernatant was subjected to CL measurement. As shown in Fig. 2, in the absence of the target DNA, CuS NPs could not be introduced, as expected, and
a
Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, P. R. China. E-mail: [email protected] b Key Laboratory of Detection Technology of Shandong Province for Tumor Marker, College of Chemistry and Chemical Engineering, Linyi University, Linyi 276005, P. R. China † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c5cc01317h
This journal is © The Royal Society of Chemistry 2015
Fig. 1 Schematic illustration of the cation-exchange-based chemiluminescence assay and its use in detecting DNA through sandwich assembly.
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Fig. 2 CL kinetic response in the presence (a) and absence (b) of 4.0 10 12 M target DNA.
Fig. 3 The CL reaction time under different conditions. Cu2+ was produced by cation-exchange with AgNO3 or an oxidation reaction with nitric acid. The concentration of the target DNA was 1.0 10 12 M.
the CL intensity was negligible (curve b). However, the CL intensity increased substantially in the presence of 4.0 10 12 M target DNA (curve a), indicating that a large amount of Cu2+ ions was produced. To further verify the feasibility of the cation-exchange reaction, inductively coupled plasma-mass spectrometry (ICP-MS) was used to detect the produced Cu2+ directly (see Table 1). At the same time, the influence of Ag+ ions on the CL reaction was investigated carefully. Fig. S4 (ESI†) showed that Ag+ showed almost no effect on the luminol–H2O2 system or the Cu2+–luminol–H2O2 system across a wide concentration range (from 4.0 10 4 M to 4.0 10 16 M) due to the relatively weak oxidation effect of AgNO3.1a So, we can conclude that the CXCLAmp strategy works well and can be used to determine the target content. The time taken for the completion of the cation-exchange reaction was compared with that of oxidation by HNO3 (0.16 M, 10 mL). The results in Fig. 3 show that both reactions could complete within a few minutes at room temperature. But the CL signal of oxidation by HNO3 was much lower when using the same concentration of target DNA. This effect may be explained because the strong acid HNO3 is unfavourable for the reaction between luminol and H2O2, which is usually carried out in a strongly basic medium. Therefore, the detection limit of this method (1.0 10 14 M, see the ESI†) is approximately 50 times lower than that of a similar sandwich CL detection reported by Zhang et al.,6a who applied strong acid and even amplified by the anodic stripping voltammetry technique. The regeneration ability is an important property for a biosensor. Unlike oxidation by nitric acid, which could destroy the structure of DNA completely, the CXCLAmp strategy can be used for the continuous detection of the target DNA after 3 or 4 regeneration cycles (see Fig. S5, ESI†) due to the highly compatible reaction conditions. Moreover, the proposed CXCLAmp strategy can also be used in the regeneration of biosensors that distinguish between antigens and antibodies as well as between carbohydrates and lectins.
On the base of feasibility study for the CXCLAmp strategy, we further decided to detect miRNA using this method, coupled with rolling circle amplification for signal amplification. The RCA reaction, which utilises DNA polymerases to generate a long single DNA strand with repeated sequences,11 was employed for introducing a large number of CuS NP tags. As shown in Fig. 4A, this RCA-amplified CXCLAmp strategy began with immobilising the capture DNA, comprising a miRNA recognition segment and a universal adapter segment. Without the target miRNA, the ligated adapter could not hybridise with the capture DNA on the MB because the melting temperature of such a double-stranded structure was below the reaction temperature (45 1C). However, in the presence of the target miRNA, the ligated adapter could link to miRNA using T4 ligase. Then, the ligated adapter could be extended through RCA when reacted with padlock DNA, T4 ligase and phi29 polymerase. The obtained long sequence, with many repeated copies, facilitates incorporating a large number of CuS NPs after hybridising with the reporter DNA–CuS NP complex. When adding AgNO3, Cu2+ was released through a cation-exchange reaction, which could catalyse a CL reaction, as described above. Thus, such a RCA– CXCLAmp strategy created a cascade of signal enhancements for the sensitive detection of miRNA. To prove the amplification effect of the RCA reaction, the CL performances under different conditions were compared, as shown in Fig. 4B. When the simple sandwich CXCLAmp strategy was applied (shown in Fig. 1), compared with the background of RCA–CXCLAmp (curve a), an obvious enhancement of the CL intensity in the presence of 1.0 10 12 M DNA could be observed (curve b). However, using the RCA–CXCLAmp strategy, a much greater enhancement of the CL intensity was observed, even in the presence of 1.0 10 14 M miRNA (curve c). The experimental results indicated that the RCA reaction could substantially amplify the CL signal for CXCLAmp detection. From the ICP-MS measurement shown in Table 1, we could see that the content of cupric ions was related to the concentration
Table 1
The total amount of Cu2+ released to solution after the cation–exchange reaction measured by ICP-MS
Concentration of DNA/RNA (M) 2+
Cu concentration for CXCLAmp (M) Cu2+ concentration for RCA–CXCLAmp (M)
Chem. Commun.
0 o0.000 o0.000
10
12
M
2.68 10 9.60 10
10 7 5
10
M
2.18 10 3.23 10
5 4
This journal is © The Royal Society of Chemistry 2015
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Fig. 6 Specificity of miRNA detection using the RCA–CXCLAmp method. The concentration of let-7a–g and let-7f each is 5 10 12 M.
Fig. 4 (A) Schematic illustration of the RCA-amplified CXCLAmp strategy and its use in the detection of miRNA; (B) the amplification effect of the RCA reaction. (a) Blank; (b) CXCLAmp strategy: the concentration of target DNA was 1.0 10 12 M; (c) RCA–CXCLAmp strategy: the concentration of target miRNA was 1.0 10 14 M.
of target DNA or miRNA. When more target DNAs or miRNAs were involved, a higher concentration of cupric ions was produced, which led to a higher CL signal. The amplification effect of the RCA reaction can also be demonstrated by comparing the content of cupric ions at the same concentrations of target DNA or miRNA. The detection capability of our RCA–CXCLAmp assay was determined using various concentrations of let-7a miRNA under optimum conditions. As shown in Fig. S11A and 11B (ESI†), the CL intensities of luminol–H2O2–Cu2+ increased with increasing let-7a concentration. The CL value exhibited a good linear relationship with the logarithm (lg) of the let-7a concentration
Fig. 5 The relative CL intensity in a log–linear correlation with the amount of let-7a in the range from 6.0 10 16 M to 5.0 10 11 M.
This journal is © The Royal Society of Chemistry 2015
over a wide dynamic range, from 6.0 10 16 M to 5.0 10 11 M (see Fig. 5). The correlation equation was I = 1.02 107 + 6.71 105 lgC (I represents the CL intensity; C is the concentration of the let-7a miRNA, n = 9, R2 = 0.9995). A detection limit of 0.17 fM (1.7 zmol) was estimated using 3s. The sensitivity of this strategy for miRNA detection could be comparable to those of most of the methods listed in Table S3 (ESI†). Moreover, after the amplification by RCA, the sensitivity of the RCA–CXCLAmp method increased approximately 100-fold compared with a simple CXCLAmp assay (see ESI†). Such a high sensitivity and wide linear range for the quantitative detection of let-7a miRNA could be attributed to the excellent performance of the CXCLAmp assay as well as the highly efficient RCA reaction. To assess the specificity of the strategy, let-7miRNA family members, which share the same lengths but differ by only 1–4 nucleotides from each other were chosen as controls. As shown in Fig. 6, all of the CL responses could be distinguished from that of the blank. The CL signal of let-7a was substantially higher than the others at the same concentration. Among these competitors, the sequences of let-7c, let-7e and let-7f have only one base mutation, indicating the excellent specificity of the developed miRNA assay. The analysis of let-7a in the 293T cell was also implemented to verify the accuracy and reliability of the method for real-world samples (see ESI†). In summary, a novel cation-exchange based chemiluminescence amplification assay was developed. Compared with the conventional CL methods, the proposed assay showed significant advantages in its low cost, nontoxic properties, ease of preparation and rapid response. The detection limit for DNA was 1.0 10 14 M using a simple sandwich strategy without any amplification process, due to the high efficiency of the cation exchange reaction, the excellent catalytic action of cupric ions for the CL reaction, and the highly compatible reaction conditions throughout the detection. Then, the RCA reaction was introduced for further enhancing the detection sensitivity. Under the optimum conditions, the detection limit for miRNA was 1.7 10 16 M with excellent specificity and could be used to detect miRNA in real-world samples. In the future, when combined with a flow-injection CL system, the performance of this strategy can be improved, making it more suitable for point-of-care detection.
Chem. Commun.
View Article Online
Communication
This work was supported by the National Natural Science Foundation of China (No. 21275003); State Key Laboratory of Microbial Technology (M2013-13).
Published on 17 March 2015. Downloaded by UNIVERSITY OF NEBRASKA on 07/04/2015 12:45:38.
Notes and references 1 (a) S. Shinsuke, N. Atsushi, A. Kenji and A. Yasuhiro, J. Am. Chem. Soc., 2005, 127, 5300; (b) H. Cui, J. Guo, N. Li and L. Liu, J. Phys. Chem. C, 2008, 112, 11319. 2 E. Marzocchi, S. Grilli, L. Della-Ciana, L. Prodi, M. Mirasoli and A. Roda, Anal. Biochem., 2008, 377, 189. 3 L. Gong, Z. Zhao, Y. Lv, S. Huan, T. Fu, X. Zhang, G. Shen and R. Yu, Chem. Commun., 2015, 51, 979. 4 C. A. Chang and H. H. Patterson, Anal. Chem., 1980, 52, 653.
Chem. Commun.
ChemComm 5 (a) Z. Li, C. Liu, Y. Fan, Y. Wang and X. Duan, Anal. Biochem., 2006, 359, 247; (b) Y. He, D. Liu, X. He and H. Cui, Chem. Commun., 2011, 47, 10692. 6 (a) C. Ding, H. Zhong and S. Zhang, Biosens. Bioelectron., 2008, 23, 1314; (b) S. Zhang, H. Zhong and C. Ding, Anal. Chem., 2008, 80, 7206. 7 D. H. Son, S. M. Hughes, Y. Yin and A. P. Alivisatos, Science, 2004, 306, 1009. 8 (a) J. Li, T. Zhang, J. Ge, Y. Yin and W. Zhong, Angew. Chem., Int. Ed., 2009, 48, 1588; (b) J. Yao, S. Schachermeyer, Y. Yin and W. Zhong, Anal. Chem., 2011, 83, 402; (c) J. Yao, X. Han, S. Zeng and W. Zhong, Anal. Chem., 2012, 84, 1645; (d) J. Li, S. Schachermeyer, Y. Wang, Y. Yin and W. Zhong, Anal. Chem., 2009, 81, 9723; (e) J. Yao, K. Flack, L. Ding and W. Zhong, Analyst, 2013, 138, 3121. ¨ur, F. M. Boldt, S. Cavaliere-Jaricot, J. Ziegler and T. Nann, 9 E. Kucu Anal. Chem., 2007, 79, 8987. 10 K. Dong, Z. Liu, Z. Li, J. Ren and X. Qu, Adv. Mater., 2013, 25, 4452. 11 W. Zhao, M. M. Ali, M. A. Brook and Y. Li, Angew. Chem., Int. Ed., 2008, 47, 6330.
This journal is © The Royal Society of Chemistry 2015
