DNA检测

Silicon-based reproducible and active surface-enhanced Raman scattering substrates for sensitive, specific, and multiplex DNA detection Z. Y. Jiang, X. X. Jiang, S. Su, X. P. Wei, S. T. Lee et al. Citation: Appl. Phys. Lett. 100, 203104 (2012); doi: 10.1063/1.3701731 View online: http://dx.doi.org/10.1063/1.3701731 View Table of Contents: http://apl.aip.org/resource/1/APPLAB/v100/i20 Published by the American Institute of Physics. Related Articles Noise spectroscopy as an equilibrium analysis tool for highly sensitive electrical biosensing Appl. Phys. Lett. 101, 093704 (2012) Ordered silicon nanocones arrays for label-free DNA quantitative analysis by surface-enhanced Raman spectroscopy Appl. Phys. Lett. 99, 153116 (2011) Optics clustered to output unique solutions: A multi-laser facility for combined single molecule and ensemble microscopy Rev. Sci. Instrum. 82, 093705 (2011) One hundred anode microchannel plate ion detector Rev. Sci. Instrum. 82, 085106 (2011) Nanoparticle-based protein detection by optical shift of a resonant microcavity Appl. Phys. Lett. 99, 073701 (2011) Additional information on Appl. Phys. Lett. Journal Homepage: http://apl.aip.org/ Journal Information: http://apl.aip.org/about/about_the_journal Top downloads: http://apl.aip.org/features/most_downloaded Information for Authors: http://apl.aip.org/authors Downloaded 11 Oct 2012 to 219.242.96.27. Redistribution subject to AIP license or copyright; see http://apl.aip.org/about/rights_and_permissions Silicon-based reproducible and active surface-enhanced Raman scattering substrates for sensitive, specific, and multiplex DNA detection Z. Y. Jiang,1 X. X. Jiang,1 S. Su,1 X. P. Wei,1 S. T. Lee,2 and Y. He1,a) 1Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Institute of Functional Nano & Soft Materials (FUNSOM), Soochow University, Suzhou 215123, China 2Center of Super-Diamond and Advanced Films (COSDAF), City University of Hong Kong, Hong Kong SAR, China and Department of Physics and Materials Science, City University of Hong Kong, Hong Kong SAR, China (Received 29 February 2012; accepted 16 March 2012; published online 14 May 2012) Silicon-based active and reproducible surface-enhanced Raman scattering (SERS) substrate, i.e., silver nanoparticles decorated-silicon wafers (AgNPs@Si), is employed for constructing high-performance sensors. Significantly, the AgNPs@Si, facilely prepared via in situ AgNPs growth on silicon wafers, features excellent SERS reproducibility and high enhancement factor. Our experiment further demonstrates such resultant silicon-based SERS substrate is efficacious for multiplex, sensitive, and specific DNA detection. In particular, single-base mismatched DNA with low concentrations is readily discriminated by using the AgNPs@Si. Moreover, the silicon-based sensor exhibits adequate multiplexing capacity, enabling unambiguous identification of the dual-target DNA detection. VC 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.3701731] Surface-enhanced Raman scattering (SERS) is one of the most powerful analytical tools for myriad sensing appli- cations due to its unique merits.1–6 For examples, compared with normal Raman signals, the SERS signals are ideally amplified 1012–1015 times when target molecules reside in the proper gap between neighboring metal nanoparticles (so called “hot spots”), allowing the acquisition of the character- istic fingerprint of low-concentration analytes. Moreover, SERS features narrow Raman bands that lead to minimal background and tremendous multiplexing capabilities. As a result, SERS has been widely utilized for wide-ranging sens- ing applications.3–6 To enable SERS techniques for practical applications, SERS substrates need to be reproducible, highly sensitive, and facilely fabricated. While solution- phase metal (e.g., silver and gold) nanoparticles employed in most ever reported multi-assay studies are well-studied, they are prone to be uncontrollably and randomly aggregated in a solution phase, yielding relatively unstable and irreproduci- ble SERS signals.7 Much effort has been devoted to improve the SERS reproducibility. Of particular note, silicon nano- structures have drawn increasingly attentions and employed for SERS applications due to many attractive properties including excellent electronic/mechanical properties, favorable biocompatibility, surface tailorability, improved multifunctoinality, as well as their compatibility with con- ventional silicon technology. Particularly, silicon nanowires (SiNWs) decorated with metal nanoparticles (e.g., AuNPs and AgNPs) have been recently developed as high- performance SERS platform with excellent reproducibility, since the metal nanoparticles are tightly immobilized by the SiNWs, effectively preventing random aggregation of the nanoparticles.8–14 As a result, such SiNWs-based SERS sub- strates have been further utilized for wide-ranging sensing applications. For instance, the gold nanoparitcle-coated SiNWs were used for multiplexed DNA detection with a detection limit of 10 pM.14 These exciting results demon- strate silicon as a promising candidate for SERS applica- tions. Nevertheless, it is worthwhile to point out that most silicon-based SERS substrates are made of SiNWs, involving relatively complicated synthetic procedures and post- treatment (e.g., metal-catalyzed vapor-liquid-solid, oxide- assisted, and HF-assisted etching growth, etc.).9 On the other hand, multiplex, sensitive, and specific DNA detection is of great demand for various chemical and biologi- cal studies.15,16 Radioactivity and fluorescence are the most established techniques for DNA detection. However, the issue of safety has been a priority concern of the radioactivity method, severely limiting its wide applications. While fluores- cence methods are capable for facile and sensitive DNA detec- tion, multiplexing detection is greatly limited due to the broad emission profiles (�50–100 nm full width at half-maximum (FWHM)) of conventional fluorophores (e.g., organic dyes).17,18 II-VI semiconductor quantum dots (QDs), serving as novel fluorescent labels with narrow spectral band (�25–40 nm fwhm), have been recently utilized for various biosensing applications.19,20 Notwithstanding, potential toxicity problems resulting from heavy metal ions (e.g., Cd ions) in the QDs have not been satisfactorily resolved and remain to be a critical issue.21,22 Therefore, it is desirable to develop new strategies for sensitive, specific, and multiplex detection of DNA. In this communication, we present a kind of silicon- based SERS-active and reproducible substrate, i.e., silver nanoparticles decorated-silicon wafers (AgNPs@Si), which is capable of facile, sensitive, and multiplex detection of DNA. AgNPs are directly in situ growth on surface of silicon wafers, producing the AgNPs@Si in very short time (�90 s). Signifi- cantly, the AgNPs@Si features excellent SERS reproducibil- ity and high enhancement factor. Our experiment further demonstrates such resultant silicon-based SERS substrate is efficacious for multiplexed DNA detection with high sensitiv- ity and specificity. a)Authors to whom correspondence should be addressed. Electronic addresses: apannale@cityu.edu.hk and yaohe@suda.edu.cn. 0003-6951/2012/100(20)/203104/4/$30.00 VC 2012 American Institute of Physics100, 203104-1 APPLIED PHYSICS LETTERS 100, 203104 (2012) Downloaded 11 Oct 2012 to 219.242.96.27. Redistribution subject to AIP license or copyright; see http://apl.aip.org/about/rights_and_permissions The AgNPs@Si is facilely synthesized via in situ growth of AgNPs on silicon wafers by an established HF-etching assisted chemical reduction method, i.e., Ag ions are reduced by Si-H bonds covered on surface of H-terminated silicon wafers (see experiment details in supporting informa- tion).9,10,26 Briefly, the cleaned silicon wafer was immersed in hydrogen fluoride (HF, 5%) solution for 30 min to achieve H-terminated silicon wafer. The silicon wafer covered by Si- H bonds was then immediately placed into a freshly prepared reduction solution containing silver nitrate (AgNO3) with slowly stirring for 90 s, achieving the AgNPs@Si. Scanning electronic microscopy (SEM) and atomic force microscopy (AFM) images of the prepared AgNPs@Si show that around 60 AgNPs with average size of �106 nm are uniformly dis- tributed on surface of silicon wafers (1 lm length� 1 lm width) (Figures 1(a) and 1(b), also see size distribution in Figure S1). Previous reports reveal that the hot spots—the gap region between a pair of AgNPs—are strongly coupled and inter-connected by the semiconducting silicon sub- strates. As a result, the AgNPs@Si yields a higher enhanced factor (EF: 8.8� 106) value compared to that of free AgNPs.8–14,23 More significantly, the AgNPs@Si possess excellent reproducibility. A large area (10 lm length� 10 lm width) AgNPs@Si dispersed with 1� 10�6 M R6G is selected for mapping test to interrogate the reproducibility of the AgNPs@Si substrate. Significantly, uniform SERS spectra recorded from 40 random spots on the substrate are observed (Figure 1(c)). Moreover, Figure 1(d) presents the correspond- ing SERS intensities of the 1517 cm�1 peak (one typical Raman peak of R6G molecules5), showing similar intensity values (�261006 3200) with relatively small standard devia- tion (12.4%). We reason that AgNPs are tightly immobilized on silicon wafers, which effectively prevents random aggre- gation of AgNPs, leading to excellent SERS reproducibility of the AgNPs@Si.7–12 To explore their utility as SERS-based DNA sensing platform, a “sandwich-type” DNA structure is further fabri- cated by using the AgNPs@Si based on established protocol (Figure 2).10,23 In brief, the AgNPs@Si is first functionalized with the thiolated single-strand capture DNA (step 1), fol- lowed by sequentially incubated with the target DNA and the reporter DNA tagged with Rhodamine 6G (R6G, one kind of commercial organic dyes, step 2). Consequently, the capture probe and the reporter probe flank the target DNA at different region, producing the sandwiched structure of cap- ture/target/reporter DNA ready for SERS detection (step 3). As a control, a single-stranded, non-cognated DNA (DNANC) that is not complementary to the capture DNA is also employed (step 20 and 30). Note that, to avoid high laser power-induced DNA damage, D2 filter is selected in our experiment, i.e., the excitation laser power (0.2 mW) equals to 1% of the original laser power (20 mW) equipped in the Raman instrument. Quantitative evaluation of the AgNPs@Si-based SERS sensor was determined by monitoring the Raman band inten- sity of R6G, covalently attached to the reporter DNA. Our analysis is based on the change of signal intensity of the prominent 1517 cm�1 Raman peak (assigned to the stretch- ing vibration of C-C bond ring mode of R6G (Refs. 24 and 25)). Figure 3(a) shows the corresponding diagnostic peaks of DNA with serial concentrations. The Raman intensity is gradually reduced along with DNA concentrations decreas- ing from 10 nM to 1 pM. Figure 3(b) shows that the Raman peak intensity initially drops approximately to 61%, 43%, 21%, or 13% of the original intensity, when the DNA con- centration is reduced to 1, 0.1, 0.01, or 0.001 nM, respec- tively. Nevertheless, the Raman intensity of 1 pM DNA (�1300) is distinctly larger than that (�30) of background, i.e., AgNP@Si without DNA modification, demonstrating high sensitivity of the AgNP@Si-based DNA sensor. To evaluate specificity of the sensor, DNANC with serial con- centrations from 10 nM to 1 pM is detected as controls. Only feeble Raman signals of R6G close to the background are observed under the same experiment conditions (blue col- umns in Figure 3(b)). The reason is that capture DNA strands are sufficiently assembled on surface of AgNPs in advance, greatly suppressing nonspecific absorbance of R6G to AgNPs.8–10 Furthermore, single-based mismatches could be discriminated due to high specificity of the prepared AgNPs@Si. Raman intensity (�1300) of the fully comple- mentary DNA is much stronger than that (�300) of the single-base mismatched targets at the same concentration FIG. 1. SEM (a) and AFM (b) images of the prepared AgNPs@Si. The AFM image is collected from a 5.0� 5.0 lm2. Raman mapping spectra (c) and corresponding Raman intensity (d) of R6G dispersed on surface of the AgNPs@Si. (kexcitation¼ 633 nm, acquisition time¼ 1 s, laser power¼ 20 mW, hole¼ 1000, slit¼ 100, grating¼ 600, and filter¼D2). FIG. 2. Schematic representation for constructing the silicon-based SERS sensor and its use for DNA detection. 203104-2 Jiang et al. Appl. Phys. Lett. 100, 203104 (2012) Downloaded 11 Oct 2012 to 219.242.96.27. Redistribution subject to AIP license or copyright; see http://apl.aip.org/about/rights_and_permissions (1 pM, Figure 3(c)). Uniform Raman spectra recorded from 25 random points on the DNA-functionalized substrates (Figure 3(d)), as well as the corresponding SERS intensities of the 1517 cm�1 peak (Figure S2) with relatively small standard deviation (13.1%), clearly suggest that the AgNPs@Si preserves stable and excellent reproducibility af- ter the DNA assembly. Moreover, small error bar obtained from at least three independent experiments in Figures 3(b) and 3(c) provides additional demonstration of the good reproducibility of the AgNPs@Si for DNA detection. Simultaneous detection of multiple analysts in a mixture without separation is a crucial requirement for the develop- ment of more effective and simpler molecular detection assays.5,9,10,14 Significantly, the AgNPs@Si allows simulta- neous anchoring of different DNA probes at the surface of AgNPs via Ag-S bonds due to its high surface-to-volume ra- tio, thus providing an opportunity for multiplexed DNA detection. In our experiment, multi-detection of specific DNA sequences through the selective capture of target strands is performed as described in Figure 4(a). Typically, two types of thiol-modified capture sequences (Pa and Pb) are mixed at equal molar ratio and co-assembled at the sur- face of AgNPs@Si (step 1). Different organic dyes (e.g., R6G and Cy3)-tagged reporter DNA strands (Ra and Rb) are employed for specifically recognizing corresponding target DNA (Ta and Tb). In absence of target DNA, only feeble background signals are observed. If one certain target DNA is added, the action of hybridization to the target sequences would lead to tremendous amplification of the Raman signals via the SERS effect (step 2a or 2b). As a result, the Raman spectra of the single-target DNA detection exhibits strong SERS signals of representative Raman peaks of R6G (a blue line, Fig. 4(b)) or Cy3 (a red line, Fig. 4(b)), respectively, with minimal spectral overlapping. Furthermore, in the case of detection of a mixture of two targets DNA, both Cy3- and R6G-tagged probes DNA are simultaneously hybridized with their corresponding targets (steps 2a and 2b), producing characteristic Raman bands of R6G and Cy3, as presented in Fig. 4(b) (black line). These results also suggest that differ- ent capture DNA assembled on the AgNPs@Si does not interfere with the specificity of the detections. To be summarized, in this study, we report AgNPs@Si as high-performance silicon-based SERS substrates for sen- sitive, specific, and multiplex DNA detection. The AgNPs@Si, facilely prepared via in situ AgNPs growth on silicon wafers, features excellent reproducibility and high enhancement factor. Significantly, systematic comparison of SERS spectra demonstrates that this new silicon-based SERS biosensor is highly sensitive and specific for DNA detection. Single-base mismatched DNA with a low concentration down to 1 pM is readily discriminated by using the AgNPs@Si. Moreover, the silicon-based sensor exhibits adequate multiplexing capacity, enabling unambiguous iden- tification of the dual-target DNA detection. Given that com- mercial silicon wafers are conveniently obtained, the high- performance silicon-based SERS sensor may serve as a potentially practical and versatile tool for myriad sensing applications. The authors would like to thank National Basic Research Program of China (973 Program 2012CB932400), NSFC (30900338, 51072116, 51132006), Research Grants Council of Hong Kong SAR, - Grant No. CityU101909, 012CB932400), and a project funded by the Priority FIG. 3. (a) SERS spectra of target DNA with different concentrations obtained from the silicon-based sensor. (b) DNA is quantitatively detected by measuring the SERS signals of R6G attached to DNA. Raman signals of DNANC and background are presented as control. (c) Detection of single- based mismatches. Raman intensity is challenged with 1 pM DNA target, mutated to either A, G, or C. (d) SERS spectra recorded from 25 random points on the substrates assembled with 10 pM target DNA. (kexcitation¼ 633 nm, laser power¼ 20 mW, acquisition time¼ 1 s, hole¼ 1000, slit¼ 100, grating¼ 600, and filter¼D2). FIG. 4. A scheme for the design of the mixed DNA-functionalized AgNPs@Si (a) and the corresponding SERS spectra for multiplex detection of target DNA (1 pM) (b). (kexcitation¼ 633 nm, laser power¼ 20 mW, acqui- sition time¼ 1 s, hole¼ 1000, slit¼ 100, grating¼ 600, and filter¼D2). 203104-3 Jiang et al. Appl. Phys. Lett. 100, 203104 (2012) Downloaded 11 Oct 2012 to 219.242.96.27. Redistribution subject to AIP license or copyright; see http://apl.aip.org/about/rights_and_permissions Academic Program Development of Jiangsu Higher Educa- tion Institutions (PAPD). 1K. Kneipp, H. Kneipp, and J. Kneipp, Acc. Chem. Res. 39, 443 (2006). 2K. Kneipp, Y. Wang, H. Kneipp, L. T. Perelman, I. Itzkan, R. R. Dasari, and M. S. Feld, Phys. Rev. Lett. 78, 1667 (1997). 3S. M. Nie and S. R. Emory, Science 275, 1102 (1997). 4J. F. Li, Y. F. Huang, Y. Ding, Z. L. Yang, S. B. Li, X. S. Zhou, F. R. Fan, W. Zhang, Z. Y. Zhou, D. Y. Wu, B. Ren, Z. L. Wang, and Z. Q. Tian, Na- ture 464, 392 (2010). 5Y. W. C. Cao, R. C. 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