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Mixed DNA-functionalized nanoparticle probes for surface-enhanced


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Mixed DNA-functionalized nanoparticle probes for surface-enhanced Raman scattering-based multiplex DNA detectionw
Zhiliang Zhang,ab Yongqiang Wen,*a Ying Ma,a Jia Luo,a Lei Jianga and Yanlin Song*a
Downloaded by McGill University on 07 June 2011 Published on 19 May 2011 on http://pubs.rsc.org | doi:10.1039/C1CC11062D

Received 23rd February 2011, Accepted 12th April 2011 DOI: 10.1039/c1cc11062d Highly stable silver nanoparticle-oligonucleotide conjugates were prepared. Based on the mixed DNA-functionalized silver nanoparticles (AgNPs), multiplex DNA detections were demonstrated successfully by SERS. Highly e?cient detection and identi?cation of speci?c analytes is essential for clinical diagnostics, environmental monitoring, safety inspection, forensics, drug testing and veterinary medicine, etc.1 Among the various detection techniques, ?uorescence is currently the most widely used method because of its high sensitivity and selectivity. Nevertheless, the inherent drawbacks of ?uorescence, such as photobleaching, narrow excitation with broad emission pro?les and peak overlapping in multiplexed experiments, hampers the application of this technology.2,3 In contrast to ?uorescence detection, surfaceenhanced Raman scattering (SERS) shows unique potential owing to its high selectivity, high sensitivity, insensitivity to quenching and, in particular, tremendous multiplexing capabilities for simultaneous target detection in one sample due to the rich molecular information and the small linewidth of vibrational Raman bands.2–6 The SERS e?ect relies on surface plasmon-enhanced optical interactions and is expected to be dramatically enhanced when Raman label molecules are localized in the interstices of nanoparticle aggregates. In recent years, with the development of nanotechnology, SERS has been increasingly investigated and great e?orts have been made to improve the sensitivity, enhance the capability for simultaneous multiple species identi?cation and develop novel e?ective assays.2–16 Despite intensive research e?orts, the progress of SERS-based theoretical and technological applications has been hampered by the di?culty of the preparation of stable and reproducible SERS substrates.4,5 So far, no completely satisfactory SERS theory has been established and SERS has yet to be translated into a widely accepted, commercially viable detection technology.2,5 Fabrication of stable SERS active substrates and development of a new detection strategy so as to fully unleash the potential of SERS are highly desired. In this manuscript, highly stable silver nanoparticle– oligonucleotide conjugates were prepared based on DNA with triple cyclic disul?de-anchoring groups. A novel SERS-based multiplex DNA detection strategy was then proposed using a sandwich assay (Scheme 1), in which mixed DNA-functionalized AgNPs 1, as well as a Raman dye and certain DNA-functionalized AgNPs 2a–c, were designed for use as the SERS probes. The multiplex DNA detection capacity was demonstrated successfully by SERS using this sandwich assay system. Our research o?ered a combined development of SERS in terms of the stability, controllability, and multiplexing capabilities, which might open up many opportunities towards the development of more practical SERS detection systems. The stability of the nanoparticle–DNA conjugates is extremely pivotal for truly practical SERS-based DNA detection systems.5 In the previous studies, it has been demonstrated by several groups and ourselves that DNA capped with cyclic disul?de can form strong polyvalent linkages with the surface of the nanoparticles and give rise to increased stability of the DNA–nanoparticle conjugates compared to capping with a monothiol group.17–19 In order to improve the controllability of SERS detection, in this work, triple cyclic disul?demodi?ed DNA molecules were grafted onto the surface of AgNPs to prepare stable SERS probes. AgNPs were used for the SERS probes because of their greater enhancement e?ect compared to other metal surfaces, and as they are simple to synthesize and produce an easily manipulated, roughened surface that is ideal for SERS analysis.8 The stability of the DNA–AgNP conjugates was evaluated by monitoring the changes in the plasmon band absorbance at 410 nm using a UV-Vis spectrophotometer under a wide range of NaCl concentration conditions. With the protection of triple-cyclic disul?de-modi?ed DNA, the AgNPs and their UV-Vis spectra are relatively stable, with only minor relative intensity changes at NaCl concentration 0.1–1.0 M (Fig. 1). In contrast, the UV-Vis spectra of the monodentate thiolconjugated AgNPs shows a rapid degradation under the same conditions (Fig. S2w). These results indicated that the triple cyclic disul?de-modi?ed DNA formed polyvalent linkages with the surface of the AgNPs, which resulted in an increased binding a?nity and much higher stability than the monothiol group provided.
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a

Beijing National Laboratory for Molecular Sciences (BNLMS), Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China. E-mail: wyq_wen@iccas.ac.cn, ylsong@iccas.ac.cn b Research Center of Analysis and Test, Shandong Polytechnic University, Jinan 250353, China w Electronic supplementary information (ESI) available: Materials, experimental details, and mechanism. See DOI: 10.1039/c1cc11062d

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Scheme 1 Schematic diagrams showing the design and operating principle of a SERS multiplex detection system. (a) A scheme of the preparation of the mixed DNA-functionalized AgNPs 1. (b) A scheme of the preparation of the Raman dye and DNA-functionalized AgNPs 2. Cyclic disul?de-modi?ed DNA and thiolated Raman reporter molecules were sequentially grafted onto the surface of the AgNPs (c). The oligonucleotide sandwich assay system for one (i), two (ii), and three (iii) target DNA molecule detection. All of the DNA probes used here are triple-cyclic disul?de-modi?ed. The reporter molecules are 4-aminothiophenol, 6-mercaptonicotinic acid and 2-mercaptopyrimidine, respectively. ‘‘T’’ represents target DNA.

Fig. 1 UV-Vis extinction spectra changes of the AgNPs conjugated with triple-cyclic disul?de-modi?ed DNA in 50 mM phosphate bu?er at various salt concentrations: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 M. All of the data were obtained after mixing the AgNPs in the solution for 48 h. The inset shows the extinction spectra of the DNA-functionalized silver nanoparticles.

The simultaneous detection of multiple analytes in a mixture without separation is a crucial requirement for the
Chem. Commun.

development of more e?ective and simpler molecular detection assays.9,12,13 Given that the high surface-to-volume ratio of AgNPs allows the accommodation of many probes at one particle, thus providing an opportunity to design a multiplex detection system with minimal optimization of the silver nanoprobes. In our experiment, the detection of speci?c DNA sequences through the selective capture of target strands by SERS nanoparticle probes using sandwich structures was performed as described in Scheme 1. In this detection system, two sets of functionalized AgNPs (1 and 2) were prepared using cyclic disul?de-modi?ed DNA (Scheme 1). In the conjugates 1, a mixed DNA layer composed of di?erent kinds of strands was used to improve the multiplexing capacity. The DNA probes used here were designed to be complementary to half of the target sequences. In addition, in the conjugates 2, a di?erent set of AgNPs were labeled with di?erent probe sequences and Raman reporter molecules, where the sequences were designed to be complementary to the other half of the target sequences. In a typical experiment, three types of probe sequences (Pa1, Pb1 and Pc1) were mixed at equal molar ratios and co-assembled at the surface of the AgNPs to form conjugate 1 (Scheme 1a). A certain probe sequence (Pa2, Pb2, or Pc2) and the corresponding thiol-containing Raman reporter molecules were sequentially grafted on the surface of AgNPs to prepare conjugates 2, respectively (Scheme 1b). In the absence of the target DNA, the nanoprobes are separated far away from each other, which results in only small background SERS signals (i.e. ‘‘o?’’ state). By adding one or more kinds of target DNA into the mixture of functionalized AgNP probes, the action of hybridization to the target sequences would result in the nanoparticles being pulled together in a state of controlled aggregation, where the electromagnetic enhancement of dyes on the surface of these nanoparticles is greatly enhanced. Thus, speci?c DNA detection can be realized by the selectively turning ‘‘on’’ o? Raman signals due to the formation of a ‘‘hot spot’’ nanostructure from the aggregation of AgNPs.5,6 With this system, single and multi-target DNA detections were examined using SERS, which display a good reproducibility of Raman signals (Fig. S3w). Fig. 2a shows the normalized SERS spectra from the three single-target detections. Studies indicated that the three SERS reporter molecules have unique SERS spectra, as well as approximately similar SERS Raman cross sections, thus allowing the detected targets to be easily distinguished from each other. As shown in Fig. 2a, the Raman spectra of the single-target detection of I, II, and III exhibit strong SERS signals at 1098 cm?1, 1435 cm?1 and 991 cm?1, respectively, with minimal spectral overlapping. Thus, they can be regarded as the characteristic signals of the three one-target DNA detections. Fig. 2b shows the SERS spectra of the multi-target detections, including the two-target and three-target detections. They all exhibited the characteristic signals of the components based on the wavenumber values. As an example, in the case of the detection of a mixture of target a (Ta) and target b (Tb), the marker bands at 1098 cm?1 and at 991 cm?1 can be clearly resolved (Fig. 2b). Thus, an unambiguous identi?cation of the three two-target detections is feasible, including discrimination from each other, as well as from the three one-target and the threetarget detections. Our research indicated that the mixed
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Downloaded by McGill University on 07 June 2011 Published on 19 May 2011 on http://pubs.rsc.org | doi:10.1039/C1CC11062D

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Downloaded by McGill University on 07 June 2011 Published on 19 May 2011 on http://pubs.rsc.org | doi:10.1039/C1CC11062D

Fig. 2 SERS spectra of the mixed DNA-functionalized silver nanoparticle probes used in the multiplexing study for (a) one- and (b) multi- component target detection. The peaks used for comparison were labeled with an asterisk. SERS spectra I, II, and III were obtained after addition of target Ta, Tb and Tc, respectively. SERS spectra IV, V, VI, and VII were obtained after addition of the mixture of target Tb/Tc, Ta/Tc, Ta/Tb, and Ta/Tb/Tc, respectively.

In order to display the detection results intuitively, a bar code diagram was generated to visualize the spectral di?erences of seven distinct one-, two-, and three-target detections. As shown in Fig. 3, the SERS central peak position can be represented by the position of the bars. It is clearly visible that the bar code signatures of the two- and three-target detections are the sum of the signatures from the corresponding one-target detection. In conclusion, in this study we not only developed the fabrication method for a stable SERS substrate, but demonstrated an e?cient multiplexed assay strategy to detect speci?c DNA in a controlled and reproducible manner using SERS. The multiplex capacity could be signi?cantly increased using mixed DNA-functionalized nanoparticle probes with respect to single-component DNA-functionalized nanoparticles. Theoretically, the multiplexing capacity when using this method could be further improved with more kinds of DNA probes grafted onto the surface of AgNPs. This SERS-based multiplex detection approach could greatly enhance the analytic e?ciency, simplify the process and reduce the amount of sample and reagent needed for analysis, as well as the time and cost of the measurement. Our strategy for probe preparation can be considered as a universal method and we expect it to be used for more practical SERS-based diagnostic analysis. The authors would like to thank the NSFC (Grant Nos. 21003132, 21073203, 21004068, 50973117, 21074139 and 20904061), 973 Program (No. 2007CB936403, 2009CB93 0404, 2011CB932303 and 2011CB808400) and CAS.

Notes and references
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DNA-modi?ed nanoparticle probes did not interfere with the speci?city of the detections.

Fig. 3 A bar code representation of the speci?c bands of the SERS spectra from di?erent single-target (I, II, and III), two-target (II + III, I + III and I + II) and three-target (I + II + III) detections.

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