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DNA-templated in situ growth of AgNPs on SWNTs: a new approach for highly sensitive SERS assay of microRNA† Jing Zheng,‡a Junhui Bai,‡a Qifeng Zhou,a Jishan Li,a Yinhui Li,a Jinfeng Yangb and Ronghua Yang*ac
DOI: 10.1039/c5cc01003a www.rsc.org/chemcomm
In this communication, we find that the single-walled carbon nanotubes (SWNTs) can demonstrate an excellent ssDNA concentration-dependent surface enhanced Raman scattering (SERS) effect after the decoration of DNA-templated in situ grown AgNPs on the surface. Inspired by this, the SWNT@AgNPs hybrid nanocomposite was employed to achieve microRNA detection which is also sensitive to crude extraction from human breast cancer cells and even patient tissues before and after chemotherapy.
Owing to their unique one-dimensional (1-D) structure, singlewalled carbon nanotubes (SWNTs) exhibit several distinctive Raman scattering features, including the radial breathing mode (RBM), the higher frequency D- (disordered) and tangential mode (G-band),1 which are sharp and obvious peaks that can easily be distinguished from fluorescence backgrounds and thus be suitable for Raman sensing.2,3 In spite of considerable progress, single SWNT typically still could not provide enough enhancement of the Raman signal and their potential as Raman tags for highly sensitive detection remains a challenge. To address this issue, a lot of efforts have been devoted to the study of the impact of different parameters and properties of the nanostructured substrates to enhance their SERS efficiency. For instance, SERS studies which are able to enhance Raman signals of SWNTs by integrating with metal nanoparticles as high as many orders of magnitude have gained particular interest.4 Deoxyribonucleic acid (DNA) has been used as an inexpensive, well-characterized, controllable, and easily adaptable material whose physical properties can be utilized to build inorganic
nanostructures, such as silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) for the SERS enhancement of SWNTs.5 As a continuation of our studies on SERS assays for biomolecules based on oligonucleotides,6,7 we are interested in investigating whether this AgNP decoration-based SERS enhancement strategy could be explored as an effective SERS assay in bioanalytical applications. As we expected, we find that seed attachment, seeded growth and DNA-templated AgNP-coated SWNT nanocomposite (SWNT@AgNPs) yielding demonstrated an excellent ssDNA concentrationdependent SERS effect, as shown in Scheme 1A. Inspired by this, we would present a new methodology by integrating our constructed ssDNA-mediated SWNT@AgNPs nanocomposites to achieve miRNA detection in this communication. As shown in Scheme 1B, hybridized double stranded DNA was conjugated on the surface of silica microbeads (SiMBs) and its affinity with SWNTs is significantly weak in the absence of target.8 Upon addition of silver ions (Ag+) and reducing agents, almost no AgNPs were coated on the surface of SWNTs, very weak D-band was observed and the G-band was also difficult to detect, thus the SERS signal remains ‘‘off’’. However, in the presence of target miRNA, competitive binding of the target forces the SWNT complex with the liberated ssDNA to act as a ‘‘nanoscaffold’’ for Ag+ to form SWNT@AgNPs on the surface of SiMB upon reduction. These ssDNA-mediated SWNT@AgNPs then showed obvious D-bands
a
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, China. E-mail: [email protected] b Department of Anesthesiology, The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, 410013, China c School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha, 410004, China † Electronic supplementary information (ESI) available: More experimental details and spectroscopic data as noted in the text. See DOI: 10.1039/c5cc01003a ‡ J. Z. and J. B. contributed equally to this work.
6552 | Chem. Commun., 2015, 51, 6552--6555
Scheme 1 Design scheme of DNA-templated in situ growth of AgNPs on SWNTs based on SERS assay for highly sensitive miRNA detection.
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and strong G-bands and the intensity was proportional to the concentration of target miRNA. Thus, this approach can be used to directly and sensitively detect the target miRNA. To the best of our knowledge, this work demonstrated the application of the SERS enhancement of DNA-mediated AgNPs-coated SWNTs in bioanalysis for the first time, which provides a promising platform for target analysis and clinic biomedical application. To fabricate SERS-active highly sensitive substrates, ssDNAmediated SWNT@AgNPs were synthesized in the solution phase using in situ Ag+ attachment and seeded growth methods. Excessive raw SWNTs were first modified by ssDNA through noncovalent binding. The z-potential increased from 13.7 mV for SWNTs to 19.5 mV for SWNT/ssDNA indicating the successful DNA binding on SWNTs. Subsequently, excess AgNO3 was added to form a SWNT/ssDNA/Ag+ complex followed by centrifugation at 14 000 rpm for 1 h to remove free Ag+, the z-potential was measured to be 3.1 mV. After reducing the SWNT/ssDNA/Ag+ complex with NaBH4, AgNPs were formed on the surface of SWNTs. The TEM image of the synthesized SWNT@AgNPs revealed that AgNPs were densely decorated on the surface of SWNT/ssDNA after seeded growth, as shown in Fig. 1A, while adding lower or higher concentrations of ssDNA solutions resulted in much different structures (Fig. S1, ESI†). The elemental composition determined by energydispersive X-ray spectroscopy (EDX) further evidenced the coexistence of C, P and Ag elements (Fig. 1B). The mass ratio of AgNPs to SWNTs in the nanocomposites was evaluated to be 13.76. Additionally, X-ray photoelectron spectroscopy (XPS) was employed to confirm the presence of AgNPs which were in situ synthesized on SWNT/ssDNA (Fig. S2, ESI†). After the growth of AgNPs on the surface of SWNT/ssDNA upon addition of AgNO3 and NaBH4, both Ag3d3/2 and Ag3d5/2 peaks at 373.8 and 368.7 eV appeared obviously. We next used AFM images to characterize the surface features of the formed AgNP aggregates on the surface of SWNT/ssDNA, which exhibited a mean height between 80 and 100 nm, being largely higher than that of the bare SWNTs
(Fig. S3, ESI†). UV-Vis spectra of SWNT@AgNPs (Fig. 1C) displayed the absorption peaks at around 420 nm, which was attributed to the surface plasmon resonance of synthesized AgNPs. Meanwhile, the digital photos of SWNT@AgNPs nanocomposite solutions synthesized by introducing ssDNA clearly showed obvious colour change (inset, Fig. 2C). It is worth noting that a continuous red-shift of the surface plasma absorption peak and change in absorption intensity appeared in the UV-Vis spectra as a function of increasing concentration of ssDNA (Fig. S4, ESI†). These collective results demonstrated that the ssDNA had actually assisted in serving as a ‘‘nanoscaffold’’ for Ag+, subsequently, formed DNA-mediated SWNT@AgNPs upon reduction. We next carefully studied the SERS enhancement of the prepared SWNT@AgNPs under varied conditions (the sequence of ssDNA is shown in Table S1, ESI†). Fig. 2 shows a set of SERS spectra of SWNT@AgNPs as a function of different concentrations of ssDNA. Either the free AgNPs (the representative TEM image is shown in Fig. S5, ESI†), free SWNTs or the SWNTs upon addition of AgNO3 and NaBH4 show very weak SERS signals by the weak interparticle plasmon coupling. However, upon addition of ssDNA to the mixture, a stronger SERS signal was demonstrated after the formation of ssDNA-mediated SWNT@AgNPs nanocomposites. The observed Raman bands mainly centered at the D-band (1348 cm 1) and the strong G-band (1605 cm 1) of SWNT. The SERS enhancement, I/I0, of the most prominent Raman peak (G-band) at 1605 cm 1 was estimated to be 20.69-fold by 300 nM ssDNA wrapping the SWNTs, where I0 and I are the SERS intensities at 1605 cm 1 in the absence and the presence of ssDNA, respectively. According to methods in the literature,6 an enhancement factor of B25 was determined which is comparable with that in the previous report.5 The inset of Fig. 3 reveals that the SERS signal dramatically increased upon increasing the ssDNA concentration under the optimal conditions (optimized concentration of NaBH4 and the growth time of AgNPs are 6 mM and 100 min, respectively, as shown in Fig. S6 and S7 (ESI†); representative SERS spectra of SWNT@AgNPs nanocomposites as a function of different concentrations of ssDNA are shown in Fig. S8, ESI†). The I/I0 value linearly
Fig. 1 Characterization of ssDNA-mediated SWNT@AgNPs nanocomposites. (A) TEM image of ssDNA-mediated SWNT@AgNPs nanocomposites. Inset: TEM image of SWNTs without ssDNA wrapping upon addition of AgNO3 and NaBH4. (B) EDX of the SWNT@AgNPs nanocomposites in the STEM pattern. (C) UV-Vis absorption spectra of SWNTs (a), free AgNPs (b) and SWNT@AgNPs nanocomposites (c). Inset: photos of (a) SWNTs, (b) free AgNPs and (c) SWNT@AgNPs nanocomposites. [SWNT] = 0.02 mg mL 1, [ssDNA] = 300 nM, [AgNO3] = 100 mM, [NaBH4] = 20 mM. The concentration of SWNTs is excessive.
Fig. 2 SERS spectra of free AgNPs (green), free SWNTs (black), SWNTs + AgNO3 + NaBH4 (blue), and (SWNTs + 300 nM ssDNA) + AgNO3 + NaBH4 (red) in 20 mM Tris-HNO3 buffer. Inset: SERS intensity enhancements of the 1605 cm 1-band of SWNTs, I/I0, plotted against the concentration of ssDNA. All error bars were obtained through the detection of six parallel samples. [SWNT] = 0.02 mg mL 1, [AgNO3] = 100 mM, [NaBH4] = 6 mM.
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Fig. 3 Performances of SERS detection of miR-21 in buffer solution. (A) SERS spectra of SWNT@AgNPs upon addition of different concentrations of miR-21. The arrow indicates the signal changes as increases in the miR-21 concentration (0, 0.01, 0.1, 0.5, 1.0, 2.0, 10, 50, 100 and 150 nM). (B) Dependence of SERS intensity enhancement of the 1605 cm 1-band, I/I0, on different concentrations of miR-21 (a), SM-21 (b), miR-141 (c), miR-143 (d) and the mixture of a, b, c, d (e) in buffer solution. Inset: I/I0 plotted against the concentration of a, b, c, d and e ranging from 0 to 2.0 nM. The measuring conditions as shown in Fig. 2. [P1] = [P4] = 400 nM. All error bars were obtained through the detection of six parallel samples.
increased with the ssDNA concentration between 0.01 and 50 nM. The limit of detection, based on 3sb/slope, where sb is the standard deviation of blank samples, was 3 pM. This result could also be improved by rational introduction of a signal amplification strategy. In order to attain a preferable separating effect and eliminate the false positive signal produced by non-target DNA or RNA, SiMB was employed to serve as an effective substrate. Since miR-21 is a potential cancer biomarker which has been identified with elevated expression levels in numerous tumour tissues, including breast, liver, ovarian, pancreatic, and brain tissues, in comparison to their normal counterparts,9 we chose it as the model target in our design. SEM images showed that the formed AgNPs were very closely aggregated on the surface of SiMBs upon addition of miR-21, and a few bright spots appeared in the image, as shown in Fig. S9A (ESI†). However, the control experiment, in which miR-21 was absent, showed no aggregates on the surface of SiMBs (inset, Fig. S9A, ESI†). We further used AFM images to characterize the surface features of the formed AgNP aggregates on SiMBs, which exhibited a mean height between 50 and 100 nm (Fig. S9B and C, ESI†), being largely higher than that of the bare SWNTs. Therefore, these special ssDNA-mediated SWNT@AgNPs have the potential to provide a significant amplification of Raman signal intensity by several orders of magnitude. We then recorded the SERS spectra of these SWNT@AgNPs nanocomposites as a function of different concentrations of miR-21. As shown in Fig. S10 (ESI†), we chose P4 as the ideal assistant probe in the subsequent experiment (P1–5, sequences are shown in Table S1, ESI†). An obvious SERS signal increase was demonstrated while increasing the complementary sequence length of the assistant probe. However, once the complementary sequence length is increased to 16 bases, the dsDNA hybridized by the capture probe and the assistant probe cannot be disassembled even in the presence of a high concentration of miR-21. Upon addition of miR-21 to the mixture, a stronger SERS signal was demonstrated for SWNT@AgNPs nanocomposites on the surface of SiMBs. As shown in Fig. 3A, the SERS enhancement, I/I0, of the
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most prominent Raman peak at 1605 cm 1 was estimated to be 23.6-fold upon addition of 100 nM miR-21, where I0 and I are the SERS intensities at 1605 cm 1 in the absence and the presence of miR-21, respectively. Fig. 3B shows that the SERS signal was proportional to the concentrations of miR-21 from 0 nM to 200 nM, establishing the quantitative detection capability of this SERS assay. The I/I0 value linearly increased with the ssDNA concentration between 0.015 and 60 nM. The limit of detection was 5 pM. These results indicate that our constructed SWNT@AgNPs nanocomposites are appropriate for highly sensitive quantification of miRNA. Additionally, Fig. 3B also shows that there is very weak SERS intensity enhancement (I/I0) in the presence of interfering miRNA (SM-21, miR-141 and miR-143) compared with the complementary target miR-21. Besides, nearly negligible SERS change was observed upon the addition of the mixture containing SM-21, miR-141 and miR-143 compared with the blank. This provides an excellent selectivity in the development of miR-21 SERS assay based on ssDNA-mediated SWNT@AgNPs nanocomposites. To investigate the feasibility of our developed SERS assay to detect miR-21 in real cell samples, we then analyzed cell lysate samples from MCF-7, HeLa cell lines and normal MCF-10A cells. The overexpression of miR-21 in MCF-7 cells has been observed previously using complicated procedures like RT-PCR or enzymatic amplification.10,11 Fig. 4A shows the SERS intensity enhancement upon the addition of different numbers of MCF-7 cells (curve a). A dramatic increase in the SERS intensity was observed as the number of cells increased from one to hundreds. The limit of detection was approximately as low as 30 cells. We also studied how SERS intensity changed upon the addition of negative control HeLa and MCF-10A cells. The results demonstrated that the SERS intensity was negligible even in the presence of 2500 HeLa cells and MCF-10A cells (curve b and curve c). Then, we mixed MCF-7 and MCF-10A cells together at different ratios of 0 : 1, 1 : 1, 5 : 1 and 10 : 1 with the total cell number fixed at 2500. The blank reaction contained MCF-10A cells only. The results provided in Fig. 4B show that miR-21 is mainly expressed in MCF-7 cells and barely expressed in MCF-10A cells. We can see from this that the intensity increment was linearly correlated with the number of MCF-7 cells.
Fig. 4 (A) Performances of SERS detection of miR-21 extracted from MCF-7 cancer cells (a), HeLa cancer cells (b) and MCF-10A cells (c). (B) Histogram showing the capacity to measure miR-21 in a mixture of MCF-7 and MCF-10A cells. The number ratios of MCF-7 and MCF-10A cells are 0 : 1, 1 : 1, 5 : 1 and 10 : 1, respectively, with a total of 2500. The standard deviations obtained by six repeated measurements are shown as the error bars. (C) Histogram showing the capacity to measure miR-21 extracted from tissues before (dark green column) and after (blue column) chemotherapy. a, b and c represent normal tissues, pericarcinous tissues and cancer tissues from patients, respectively. The measuring conditions as shown in Fig. 3.
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Therefore, it can be concluded that our assay has the potential for application to cell extracts along with high sensitivity and a wide dynamic range. Differential expressions of certain miRNA have been shown to be accurate predictors for a patient’s overall prognosis.12 To investigate the applicability of this approach in clinical diagnosis, we performed this assay on crude extracts of normal tissues, breast pericarcinous tissues and breast cancer tissues (Fig. S11, ESI†). Signal intensities of the pericarcinous tissue samples were slightly higher than those of the normal tissues, but those of the cancer samples were significantly higher than those of pericarcinous tissue samples, as shown in Fig. S12 and S13 (ESI†). These results demonstrate that our assay holds great promise for cancer diagnosis with great selectivity and accuracy. Furthermore, we performed this assay on crude extracts of breast cancer tissues from patients before and after chemotherapy, as shown in Fig. 4C. The results of three cases show that a significant reduction of miR-21 was observed after treating with chemotherapy, which is coincident with the previous report and the applicability of this approach in clinical diagnosis is demonstrated.13 In summary, we have developed a new ssDNA-mediated SWNT@AgNPs nanocomposite-based SERS assay to achieve miRNA detection via in situ growth of AgNPs on SWNTs, and further applied it for sensitive and selective measurement in cell lysate and even breast cancer tissues before and after chemotherapy. Compared with the previous report, this work demonstrated the application of the SERS enhancement of SWNT@AgNPs in bioanalysis for the first time, and the concept can easily be extended to construct a series of probes for sensing different kinds of biomolecules. Moreover, enzymatic amplification strategies etc. could also be employed to enable our constructed platform to achieve detection of ultra-sensitive
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biomolecules in future work. Given the low cost, high sensitivity, and excellent generality, we anticipate that this approach might open up new opportunities for the development of new bioanalytical and biomedical application. This work was financially supported by the National Natural Science Foundation of China (21405038, 21135001, 21305036), the Foundation for Innovative Research Groups of NSFC (21221003), the ‘‘973’’ National Key Basic Research Program (2011CB91100-0), and the Fundamental Research Funds for the Central Universities.
Notes and references 1 A. M. Rao, E. Richter, S. Bandow, B. Chase, P. C. Eklund, K. A. Williams, S. Fang, K. R. Subbaswamy, M. Menon and M. S. Dresselhaus, Science, 1997, 275, 187. 2 Z. Chen, S. M. Tabakman, A. P. Goodwin, M. G. Kattah, D. Daranciang, X. Wang, G. Zhang, X. Li, Z. Liu and H. J. Dai, Nat. Biotechnol., 2008, 26, 1285. 3 D. A. Heller, S. Baik, T. E. Eurell and M. S. Strano, Adv. Mater., 2005, 17, 2793. 4 Y. C. Chen, R. J. Young, J. V. Macpherson and N. R. Wilson, J. Phys. Chem. C, 2007, 111, 16167. 5 X. J. Wang, C. Wang, L. Cheng, S. T. Lee and Z. Liu, J. Am. Chem. Soc., 2012, 134, 7414. 6 J. Zheng, A. L. Jiao, R. H. Yang and W. H. Tan, J. Am. Chem. Soc., 2012, 134, 19957. 7 J. Zheng, Y. P. Hu, J. H. Bai, J. S. Li and R. H. Yang, Anal. Chem., 2014, 86, 2205. 8 R. H. Yang, J. Y. Jin, Z. W. Tang, Y. R. Wu, Z. Zhu and W. H. Tan, J. Am. Chem. Soc., 2008, 130, 8351. 9 S. Zhu, H. Wu, F. Wu, D. Nie, S. Sheng and Y. Y. Mo, Cell Res., 2008, 18, 350. 10 K. A. Cissell, Y. Rahimi, S. Shrestha, E. A. Hunt and S. K. Deo, Anal. Chem., 2008, 80, 2319. 11 J. Lu, G. Getz, E. A. Miska, J. Lamb, R. H. Mak, H. R. Horvitz and T. R. Golub, Nature, 2005, 435, 834. 12 A. Esquela-Kerscher and F. J. Slack, Nat. Rev. Cancer, 2006, 6, 259. 13 M. V. Blagosklonny, Cell Death Differ., 2005, 12, 592.
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DNA-templated in situ growth of AgNPs on SWNTs: a new approach for highly sensitive SERS assay of microRNA† Jing Zheng,‡a Junhui Bai,‡a Qifeng Zhou,a Jishan Li,a Yinhui Li,a Jinfeng Yangb and Ronghua Yang*ac
DOI: 10.1039/c5cc01003a www.rsc.org/chemcomm
In this communication, we find that the single-walled carbon nanotubes (SWNTs) can demonstrate an excellent ssDNA concentration-dependent surface enhanced Raman scattering (SERS) effect after the decoration of DNA-templated in situ grown AgNPs on the surface. Inspired by this, the SWNT@AgNPs hybrid nanocomposite was employed to achieve microRNA detection which is also sensitive to crude extraction from human breast cancer cells and even patient tissues before and after chemotherapy.
Owing to their unique one-dimensional (1-D) structure, singlewalled carbon nanotubes (SWNTs) exhibit several distinctive Raman scattering features, including the radial breathing mode (RBM), the higher frequency D- (disordered) and tangential mode (G-band),1 which are sharp and obvious peaks that can easily be distinguished from fluorescence backgrounds and thus be suitable for Raman sensing.2,3 In spite of considerable progress, single SWNT typically still could not provide enough enhancement of the Raman signal and their potential as Raman tags for highly sensitive detection remains a challenge. To address this issue, a lot of efforts have been devoted to the study of the impact of different parameters and properties of the nanostructured substrates to enhance their SERS efficiency. For instance, SERS studies which are able to enhance Raman signals of SWNTs by integrating with metal nanoparticles as high as many orders of magnitude have gained particular interest.4 Deoxyribonucleic acid (DNA) has been used as an inexpensive, well-characterized, controllable, and easily adaptable material whose physical properties can be utilized to build inorganic
nanostructures, such as silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) for the SERS enhancement of SWNTs.5 As a continuation of our studies on SERS assays for biomolecules based on oligonucleotides,6,7 we are interested in investigating whether this AgNP decoration-based SERS enhancement strategy could be explored as an effective SERS assay in bioanalytical applications. As we expected, we find that seed attachment, seeded growth and DNA-templated AgNP-coated SWNT nanocomposite (SWNT@AgNPs) yielding demonstrated an excellent ssDNA concentrationdependent SERS effect, as shown in Scheme 1A. Inspired by this, we would present a new methodology by integrating our constructed ssDNA-mediated SWNT@AgNPs nanocomposites to achieve miRNA detection in this communication. As shown in Scheme 1B, hybridized double stranded DNA was conjugated on the surface of silica microbeads (SiMBs) and its affinity with SWNTs is significantly weak in the absence of target.8 Upon addition of silver ions (Ag+) and reducing agents, almost no AgNPs were coated on the surface of SWNTs, very weak D-band was observed and the G-band was also difficult to detect, thus the SERS signal remains ‘‘off’’. However, in the presence of target miRNA, competitive binding of the target forces the SWNT complex with the liberated ssDNA to act as a ‘‘nanoscaffold’’ for Ag+ to form SWNT@AgNPs on the surface of SiMB upon reduction. These ssDNA-mediated SWNT@AgNPs then showed obvious D-bands
a
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, China. E-mail: [email protected] b Department of Anesthesiology, The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, 410013, China c School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha, 410004, China † Electronic supplementary information (ESI) available: More experimental details and spectroscopic data as noted in the text. See DOI: 10.1039/c5cc01003a ‡ J. Z. and J. B. contributed equally to this work.
6552 | Chem. Commun., 2015, 51, 6552--6555
Scheme 1 Design scheme of DNA-templated in situ growth of AgNPs on SWNTs based on SERS assay for highly sensitive miRNA detection.
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and strong G-bands and the intensity was proportional to the concentration of target miRNA. Thus, this approach can be used to directly and sensitively detect the target miRNA. To the best of our knowledge, this work demonstrated the application of the SERS enhancement of DNA-mediated AgNPs-coated SWNTs in bioanalysis for the first time, which provides a promising platform for target analysis and clinic biomedical application. To fabricate SERS-active highly sensitive substrates, ssDNAmediated SWNT@AgNPs were synthesized in the solution phase using in situ Ag+ attachment and seeded growth methods. Excessive raw SWNTs were first modified by ssDNA through noncovalent binding. The z-potential increased from 13.7 mV for SWNTs to 19.5 mV for SWNT/ssDNA indicating the successful DNA binding on SWNTs. Subsequently, excess AgNO3 was added to form a SWNT/ssDNA/Ag+ complex followed by centrifugation at 14 000 rpm for 1 h to remove free Ag+, the z-potential was measured to be 3.1 mV. After reducing the SWNT/ssDNA/Ag+ complex with NaBH4, AgNPs were formed on the surface of SWNTs. The TEM image of the synthesized SWNT@AgNPs revealed that AgNPs were densely decorated on the surface of SWNT/ssDNA after seeded growth, as shown in Fig. 1A, while adding lower or higher concentrations of ssDNA solutions resulted in much different structures (Fig. S1, ESI†). The elemental composition determined by energydispersive X-ray spectroscopy (EDX) further evidenced the coexistence of C, P and Ag elements (Fig. 1B). The mass ratio of AgNPs to SWNTs in the nanocomposites was evaluated to be 13.76. Additionally, X-ray photoelectron spectroscopy (XPS) was employed to confirm the presence of AgNPs which were in situ synthesized on SWNT/ssDNA (Fig. S2, ESI†). After the growth of AgNPs on the surface of SWNT/ssDNA upon addition of AgNO3 and NaBH4, both Ag3d3/2 and Ag3d5/2 peaks at 373.8 and 368.7 eV appeared obviously. We next used AFM images to characterize the surface features of the formed AgNP aggregates on the surface of SWNT/ssDNA, which exhibited a mean height between 80 and 100 nm, being largely higher than that of the bare SWNTs
(Fig. S3, ESI†). UV-Vis spectra of SWNT@AgNPs (Fig. 1C) displayed the absorption peaks at around 420 nm, which was attributed to the surface plasmon resonance of synthesized AgNPs. Meanwhile, the digital photos of SWNT@AgNPs nanocomposite solutions synthesized by introducing ssDNA clearly showed obvious colour change (inset, Fig. 2C). It is worth noting that a continuous red-shift of the surface plasma absorption peak and change in absorption intensity appeared in the UV-Vis spectra as a function of increasing concentration of ssDNA (Fig. S4, ESI†). These collective results demonstrated that the ssDNA had actually assisted in serving as a ‘‘nanoscaffold’’ for Ag+, subsequently, formed DNA-mediated SWNT@AgNPs upon reduction. We next carefully studied the SERS enhancement of the prepared SWNT@AgNPs under varied conditions (the sequence of ssDNA is shown in Table S1, ESI†). Fig. 2 shows a set of SERS spectra of SWNT@AgNPs as a function of different concentrations of ssDNA. Either the free AgNPs (the representative TEM image is shown in Fig. S5, ESI†), free SWNTs or the SWNTs upon addition of AgNO3 and NaBH4 show very weak SERS signals by the weak interparticle plasmon coupling. However, upon addition of ssDNA to the mixture, a stronger SERS signal was demonstrated after the formation of ssDNA-mediated SWNT@AgNPs nanocomposites. The observed Raman bands mainly centered at the D-band (1348 cm 1) and the strong G-band (1605 cm 1) of SWNT. The SERS enhancement, I/I0, of the most prominent Raman peak (G-band) at 1605 cm 1 was estimated to be 20.69-fold by 300 nM ssDNA wrapping the SWNTs, where I0 and I are the SERS intensities at 1605 cm 1 in the absence and the presence of ssDNA, respectively. According to methods in the literature,6 an enhancement factor of B25 was determined which is comparable with that in the previous report.5 The inset of Fig. 3 reveals that the SERS signal dramatically increased upon increasing the ssDNA concentration under the optimal conditions (optimized concentration of NaBH4 and the growth time of AgNPs are 6 mM and 100 min, respectively, as shown in Fig. S6 and S7 (ESI†); representative SERS spectra of SWNT@AgNPs nanocomposites as a function of different concentrations of ssDNA are shown in Fig. S8, ESI†). The I/I0 value linearly
Fig. 1 Characterization of ssDNA-mediated SWNT@AgNPs nanocomposites. (A) TEM image of ssDNA-mediated SWNT@AgNPs nanocomposites. Inset: TEM image of SWNTs without ssDNA wrapping upon addition of AgNO3 and NaBH4. (B) EDX of the SWNT@AgNPs nanocomposites in the STEM pattern. (C) UV-Vis absorption spectra of SWNTs (a), free AgNPs (b) and SWNT@AgNPs nanocomposites (c). Inset: photos of (a) SWNTs, (b) free AgNPs and (c) SWNT@AgNPs nanocomposites. [SWNT] = 0.02 mg mL 1, [ssDNA] = 300 nM, [AgNO3] = 100 mM, [NaBH4] = 20 mM. The concentration of SWNTs is excessive.
Fig. 2 SERS spectra of free AgNPs (green), free SWNTs (black), SWNTs + AgNO3 + NaBH4 (blue), and (SWNTs + 300 nM ssDNA) + AgNO3 + NaBH4 (red) in 20 mM Tris-HNO3 buffer. Inset: SERS intensity enhancements of the 1605 cm 1-band of SWNTs, I/I0, plotted against the concentration of ssDNA. All error bars were obtained through the detection of six parallel samples. [SWNT] = 0.02 mg mL 1, [AgNO3] = 100 mM, [NaBH4] = 6 mM.
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Fig. 3 Performances of SERS detection of miR-21 in buffer solution. (A) SERS spectra of SWNT@AgNPs upon addition of different concentrations of miR-21. The arrow indicates the signal changes as increases in the miR-21 concentration (0, 0.01, 0.1, 0.5, 1.0, 2.0, 10, 50, 100 and 150 nM). (B) Dependence of SERS intensity enhancement of the 1605 cm 1-band, I/I0, on different concentrations of miR-21 (a), SM-21 (b), miR-141 (c), miR-143 (d) and the mixture of a, b, c, d (e) in buffer solution. Inset: I/I0 plotted against the concentration of a, b, c, d and e ranging from 0 to 2.0 nM. The measuring conditions as shown in Fig. 2. [P1] = [P4] = 400 nM. All error bars were obtained through the detection of six parallel samples.
increased with the ssDNA concentration between 0.01 and 50 nM. The limit of detection, based on 3sb/slope, where sb is the standard deviation of blank samples, was 3 pM. This result could also be improved by rational introduction of a signal amplification strategy. In order to attain a preferable separating effect and eliminate the false positive signal produced by non-target DNA or RNA, SiMB was employed to serve as an effective substrate. Since miR-21 is a potential cancer biomarker which has been identified with elevated expression levels in numerous tumour tissues, including breast, liver, ovarian, pancreatic, and brain tissues, in comparison to their normal counterparts,9 we chose it as the model target in our design. SEM images showed that the formed AgNPs were very closely aggregated on the surface of SiMBs upon addition of miR-21, and a few bright spots appeared in the image, as shown in Fig. S9A (ESI†). However, the control experiment, in which miR-21 was absent, showed no aggregates on the surface of SiMBs (inset, Fig. S9A, ESI†). We further used AFM images to characterize the surface features of the formed AgNP aggregates on SiMBs, which exhibited a mean height between 50 and 100 nm (Fig. S9B and C, ESI†), being largely higher than that of the bare SWNTs. Therefore, these special ssDNA-mediated SWNT@AgNPs have the potential to provide a significant amplification of Raman signal intensity by several orders of magnitude. We then recorded the SERS spectra of these SWNT@AgNPs nanocomposites as a function of different concentrations of miR-21. As shown in Fig. S10 (ESI†), we chose P4 as the ideal assistant probe in the subsequent experiment (P1–5, sequences are shown in Table S1, ESI†). An obvious SERS signal increase was demonstrated while increasing the complementary sequence length of the assistant probe. However, once the complementary sequence length is increased to 16 bases, the dsDNA hybridized by the capture probe and the assistant probe cannot be disassembled even in the presence of a high concentration of miR-21. Upon addition of miR-21 to the mixture, a stronger SERS signal was demonstrated for SWNT@AgNPs nanocomposites on the surface of SiMBs. As shown in Fig. 3A, the SERS enhancement, I/I0, of the
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most prominent Raman peak at 1605 cm 1 was estimated to be 23.6-fold upon addition of 100 nM miR-21, where I0 and I are the SERS intensities at 1605 cm 1 in the absence and the presence of miR-21, respectively. Fig. 3B shows that the SERS signal was proportional to the concentrations of miR-21 from 0 nM to 200 nM, establishing the quantitative detection capability of this SERS assay. The I/I0 value linearly increased with the ssDNA concentration between 0.015 and 60 nM. The limit of detection was 5 pM. These results indicate that our constructed SWNT@AgNPs nanocomposites are appropriate for highly sensitive quantification of miRNA. Additionally, Fig. 3B also shows that there is very weak SERS intensity enhancement (I/I0) in the presence of interfering miRNA (SM-21, miR-141 and miR-143) compared with the complementary target miR-21. Besides, nearly negligible SERS change was observed upon the addition of the mixture containing SM-21, miR-141 and miR-143 compared with the blank. This provides an excellent selectivity in the development of miR-21 SERS assay based on ssDNA-mediated SWNT@AgNPs nanocomposites. To investigate the feasibility of our developed SERS assay to detect miR-21 in real cell samples, we then analyzed cell lysate samples from MCF-7, HeLa cell lines and normal MCF-10A cells. The overexpression of miR-21 in MCF-7 cells has been observed previously using complicated procedures like RT-PCR or enzymatic amplification.10,11 Fig. 4A shows the SERS intensity enhancement upon the addition of different numbers of MCF-7 cells (curve a). A dramatic increase in the SERS intensity was observed as the number of cells increased from one to hundreds. The limit of detection was approximately as low as 30 cells. We also studied how SERS intensity changed upon the addition of negative control HeLa and MCF-10A cells. The results demonstrated that the SERS intensity was negligible even in the presence of 2500 HeLa cells and MCF-10A cells (curve b and curve c). Then, we mixed MCF-7 and MCF-10A cells together at different ratios of 0 : 1, 1 : 1, 5 : 1 and 10 : 1 with the total cell number fixed at 2500. The blank reaction contained MCF-10A cells only. The results provided in Fig. 4B show that miR-21 is mainly expressed in MCF-7 cells and barely expressed in MCF-10A cells. We can see from this that the intensity increment was linearly correlated with the number of MCF-7 cells.
Fig. 4 (A) Performances of SERS detection of miR-21 extracted from MCF-7 cancer cells (a), HeLa cancer cells (b) and MCF-10A cells (c). (B) Histogram showing the capacity to measure miR-21 in a mixture of MCF-7 and MCF-10A cells. The number ratios of MCF-7 and MCF-10A cells are 0 : 1, 1 : 1, 5 : 1 and 10 : 1, respectively, with a total of 2500. The standard deviations obtained by six repeated measurements are shown as the error bars. (C) Histogram showing the capacity to measure miR-21 extracted from tissues before (dark green column) and after (blue column) chemotherapy. a, b and c represent normal tissues, pericarcinous tissues and cancer tissues from patients, respectively. The measuring conditions as shown in Fig. 3.
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Therefore, it can be concluded that our assay has the potential for application to cell extracts along with high sensitivity and a wide dynamic range. Differential expressions of certain miRNA have been shown to be accurate predictors for a patient’s overall prognosis.12 To investigate the applicability of this approach in clinical diagnosis, we performed this assay on crude extracts of normal tissues, breast pericarcinous tissues and breast cancer tissues (Fig. S11, ESI†). Signal intensities of the pericarcinous tissue samples were slightly higher than those of the normal tissues, but those of the cancer samples were significantly higher than those of pericarcinous tissue samples, as shown in Fig. S12 and S13 (ESI†). These results demonstrate that our assay holds great promise for cancer diagnosis with great selectivity and accuracy. Furthermore, we performed this assay on crude extracts of breast cancer tissues from patients before and after chemotherapy, as shown in Fig. 4C. The results of three cases show that a significant reduction of miR-21 was observed after treating with chemotherapy, which is coincident with the previous report and the applicability of this approach in clinical diagnosis is demonstrated.13 In summary, we have developed a new ssDNA-mediated SWNT@AgNPs nanocomposite-based SERS assay to achieve miRNA detection via in situ growth of AgNPs on SWNTs, and further applied it for sensitive and selective measurement in cell lysate and even breast cancer tissues before and after chemotherapy. Compared with the previous report, this work demonstrated the application of the SERS enhancement of SWNT@AgNPs in bioanalysis for the first time, and the concept can easily be extended to construct a series of probes for sensing different kinds of biomolecules. Moreover, enzymatic amplification strategies etc. could also be employed to enable our constructed platform to achieve detection of ultra-sensitive
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biomolecules in future work. Given the low cost, high sensitivity, and excellent generality, we anticipate that this approach might open up new opportunities for the development of new bioanalytical and biomedical application. This work was financially supported by the National Natural Science Foundation of China (21405038, 21135001, 21305036), the Foundation for Innovative Research Groups of NSFC (21221003), the ‘‘973’’ National Key Basic Research Program (2011CB91100-0), and the Fundamental Research Funds for the Central Universities.
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