LETTERS
Control of enhanced Raman scattering using a DNA-based assembly process of dye-coded nanoparticles DUNCAN GRAHAM*, DAVID G. THOMPSON, W. EWEN SMITH AND KAREN FAULDS Centre for Molecular Nanometrology, WestCHEM, Department of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow, G1 1XL, UK *e-mail:
[email protected]
Published online: 11 July 2008; doi:10.1038/nnano.2008.189
Enhanced Raman scattering from metal surfaces has been investigated for over 30 years1. Silver surfaces are known to produce a large effect, and this can be maximized by producing a roughened surface, which can be achieved by the aggregation of silver nanoparticles2–4. However, an approach to control this aggregation, in particular through the interaction of biological molecules such as DNA, has not been reported. Here we show the selective turning on of the surface enhanced resonance Raman scattering5 effect on dye-coded, DNAfunctionalized, silver nanoparticles through a targetdependent, sequence-specific DNA hybridization assembly that exploits the electromagnetic enhancement mechanism for the scattering. Dye-coded nanoparticles that do not undergo hybridization experience no enhancement and hence do not give surface enhanced resonance Raman scattering. This is due to the massive difference in enhancement from nanoparticle assemblies compared with individual nanoparticles. The electromagnetic enhancement is the dominant effect and, coupled with an understanding of the surface chemistry, allows surface enhanced resonance Raman scattering nanosensors to be designed based on a natural biological recognition process. Surface enhanced Raman scattering (SERS) was first reported in 1974 when the enhanced Raman scattering of pyridine adsorbed on the surface of a silver electrode was observed1. Since then, many studies have gone on to investigate the range of surfaces and the phenomenon itself. The enhancement can be further increased by the incorporation of a coloured moiety that provides an additional resonance contribution and results in what is known as surface enhanced resonance Raman scattering (SERRS)5. It has been shown that silver has a greater enhancement effect than other metal surfaces such as gold6, and a convenient form of an enhancing silver surface is composed of silver nanoparticles. Silver nanoparticles are simple to synthesize and produce an easily manipulated roughened surface ideal for SERRS analysis2. To obtain maximum enhancement of the Raman scattering from silver nanoparticles, the monodispersed colloidal suspension needs to be aggregated into discrete clusters, which then give rise to the massive enhancement of Raman scattering reported in a number of seminal papers1,3,5,7. One of the issues of enhanced Raman scattering from silver nanoparticles has been 548
the control of the assembly of the nanoparticles to turn on the enhancement in a controlled and reproducible manner. Many approaches have been investigated in an attempt to overcome this such as the use of surface charge altering agents including poly(L-lysine)9, spermine hydrochloride10, sodium chloride11 and nitric acid12. However, all of these approaches permanently turn on the enhancement of the Raman scattering and are dependent on the addition of an external reagent rather than a biological target molecule of interest. Assembly of metallic nanoparticles has been extensively studied by a number of different groups, and gold has been predominantly investigated because of its stability and its ability to indicate the presence or absence of particular biological species through a shift in the plasmon resonance band13–15. These studies have clearly shown how the plasmon band of the nanoparticles changes through interaction, as the nanoparticles have drawn closer together through an assembly process often controlled through biological recognition using species such as DNA or antibody –antigen interactions. In a recent study it was shown how a silver nanoparticle could be used to enhance a fluorophore attached to a metal surface using DNA hybridization to place the nanoparticle in the right environment for enhancement16. This is similar to the work of Mirkin in that a surface-bound species can be enhanced through the use of an additional enhancing surface such as silver or silver nanoparticles17. In this study we report the ability to control the enhancement of Raman scattering in suspension from silver nanoparticles coded with a Raman active dye through controlled assembly formation using DNA hybridization. This approach differs from those previously reported in that functionalized nanoparticles are used and a signal is only achieved when the molecular recognition event of DNA hybridization takes place. Two mechanisms are thought to be responsible for the enhancement of Raman scattering—chemical and electromagnetic. Here we focus on controlling electromagnetic enhancement through interaction of the nanoparticles. The key to this has been an understanding of the chemistry involved in the functionalization of silver surfaces and also the synthesis of particular Raman active dyes18 for use with the silver nanoparticles when functionalized with oligonucleotide probe sequences. Our approach to coding silver nanoparticles with a specific Raman tag and the control of their assembly through hybridization is outlined in Fig. 1a, b. nature nanotechnology | VOL 3 | SEPTEMBER 2008 | www.nature.com/naturenanotechnology
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LETTERS V I
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The secret to maximizing enhancement from Raman scattering molecules on nanoparticles is to provide a state of aggregation of the silver nanoparticles and to place the Raman scattering molecule in the interstices of the assembled nanoparticles allowing it to experience the maximum enhancement. In order to do this, we have pre-functionalized silver nanoparticles in the following way. Initially, a multifunctionalized SERRS active dye was synthesized. It comprised the chromophore in the form of an azo group, a surface complexing group—benzotriazole—and a negatively charged phenolic group to prevent non-specific electrostatic aggregation of nanoparticles. This was added to the nanoparticles in a concentration in excess of that required to form a monolayer through complexation to the surface. A 50 -thiolated oligonucleotide probe containing a ten-adenine-base tether was added to the silver nanoparticles in an analogous method to that previously reported for gold nanoparticles19. The ratio of oligonucleotide to dye was 1:1. Two solutions of silver nanoparticles were prepared using this approach. Each contained different probe sequences but the same dye as the Raman tag for the nanoparticles (Fig. 1b). The functionalized coded nanoparticles were investigated for their SERRS response using different wavelengths (406 nm, 514 nm, 532 nm and 632 nm), and it was found that at all wavelengths there was a small residual background SERRS signal but that 532 nm laser excitation had the lowest signal-to-background ratio. The extinction spectrum of these nanoparticles was also obtained and is shown in Fig. 2a. It is clear from the comparison between the non-functionalized and functionalized nanoparticles that there is a slight red shift of 17 nm. However, it can clearly be seen that there is no appreciable aggregation of these nanoparticles following functionalization. The preparation procedure also resulted in the suspended
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Figure 1 Schematic representation of the synthesis of Raman dye-functionalized DNA silver nanoparticle conjugates. a, Silver nanoparticles (I) were synthesized using a previously reported citrate reduction method2. 3,5-Dimethoxy-4-(60 -azobenzotriazolyl)phenol (II) (ref. 18) was added at a concentration sufficient to ensure there was a monolayer of the dye on the nanoparticles (III). 50 -thiol-functionalized oligonucleotide probe sequences (IV) were then added to a final concentration equivalent to that of the dye to form the dye/oligonucleotide – nanoparticles conjugates (V). A period of curing with a DNA hybridization buffer followed by centrifugation removed any unreacted dye or oligonucleotides. b, An oligonucleotide sandwich assay using Raman dye-functionalized DNA – silver nanoparticle conjugates. Two batches of conjugates were functionalized with different, non-complementary probe sequences (1, 2). On addition of a target oligonucleotide complementary to both sequences 1 and 2, hybridization of the sequences occurs and results in aggregation of the nanoparticles.
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Figure 2 UV-vis analysis of dye-coded DNA-functionalized silver nanoparticles. a, Extinction spectra of silver nanoparticles (1) (l max ¼ 400 nm), Raman dye- and DNA-functionalized silver nanoparticles (2) (l max ¼ 417 nm) and Raman dye- and DNA-functionalized silver nanoparticles hybridized to a complementary target sequence in a sandwich assay for 1 h (3) (l max ¼ 550 nm). b, Melting transitions of the nanoparticle conjugates measured at 417 nm (probe sequences used: 50 (10 A)-TCTCAACTCGTA and 50 (10 A)-CGCATTCAGGAT) hybridized to a fully complementary sequence 50 -TACGAGTTGAGAATCCTGAATGCG (I) (T m ¼ 50.27 8 C), a sequence containing a mismatch at the centre of the target oligonucleotide 50 -TACGAGTTGAGACTCCTGAATGCG (II) (T m ¼ 48.07 8 C), and a sequence containing a mismatch at the 30 -terminus 50 -TACGAGTTGAGAATCCTGAATGCT(III) (T m ¼ 46.27 8 C). The concentration of nanoparticle conjugates used was 10 pM, and target oligonucleotide concentration was 2.5 nM.
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LETTERS
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Figure 3 SERRS spectra of DNA-functionalized Raman dye-coded silver nanoparticles. a, Spectra after being exposed to either a fully complementary target 50 -TACGAGAGAATCCTGAATGCG (1) or half complementary target 50 -TACGAGTTGAGACGCATTGAGGAT (2) for 60 min. b, Change in intensity at 1,368 cm21 over 60 min using 5 pM of each nanoparticle conjugate (probe sequences used are the same as in Fig. 2). The target concentration was 1.25 nM. The SERRS spectra were taken using an Avalon spectrometer, with 532 nm laser wavelength, 3 s scan time and 50% power.
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functionalized nanoparticles being in buffer conditions appropriate for DNA hybridization—0.3 M sodium chloride and 10 mM phosphate buffer at pH 7. The functionalized nanoparticles are stable for over 6 months in these conditions. The surface coverage of the oligonucleotide on the nanoparticles was determined to be 35.2+1.9 pmol cm22 using a previously published method20. For comparison, silver nanoparticles were also functionalized solely with oligonucleotide, and their surface coverage was found to be 95.1+4.4 pmol cm22, indicating that there was a slight excess of dye on the surface. The next step in this procedure was the controlled assembly of nano-aggregates through DNA hybridization. Addition of a target complementary to the two probe sequences resulted in an appreciable colour change of the nanoparticles, from yellow to green-blue, as can be seen visually and also in the extinction spectrum (Fig. 2a). This indicates that aggregation has taken place and that the silver nanoparticles are now in a state of aggregation. The assembled nanoparticles were heated and cooled to investigate the sequence discrimination effect of these silver conjugates. The melting curves as measured at the plasmon maximum of the silver nanoparticles are shown in Fig. 2b. These curves indicate that the functionalized nanoparticles display the same sharp melting transition reported for other oligonucleotide nanoparticle conjugates19 as well as the easy discrimination between a fully complementary target and containing a single base mismatch. This is an important aspect of these conjugates, proving that they are still capable of undergoing sequence-specific hybridization, and in a highly specific manner, as expected from oligonucleotide-functionalized silver or gold nanoparticles. The SERRS spectra and spectral intensity of the nanoparticle conjugates when exposed to a target oligonucleotide were then analysed in a DNA sandwich assay identical to that used for extinction analysis. When the target oligonucleotide was added to the functionalized DNA silver nanoparticle conjugates the previously reported spectrum of 3,5-dimethoxy-4-(60 -azobenzotriazolyl) phenol18 is seen to drastically increase in intensity due to the aggregation caused by the target (Fig. 3a). This change is sequence-specific and will not occur when the target does not have the necessary degree of complementarity. In addition,
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Figure 4 Selective enhancement of specific Raman signals through DNA hybridization. The assay consisted of three different conjugates. Each had a different, non-complementary oligonucleotide (sequence A: 50 -AAAAAAAAAATACAGCACG; sequence B: 50 -AAAAAAAAAATCTCAACTC; sequence C: 50 -AAAAAAAAAAGGACTACCT). Conjugates A and B were functionalized with dye 1, denoted in blue, and conjugate C was functionalized dye 2, denoted by red. These three conjugates were mixed together at 30 pM final concentration (background shown at the top). When a target complementary to A and B (50 -CGTGCTGTAGAGTTGAGA) is added, only dye 1 is enhanced (bottom left), and when a target complementary to B and C (50 -AGGTAGTCCGAGTTGAGA) is added, the spectrum for dye 1 and dye 2 is enhanced (bottom right). Spectra were taken after 20 min of hybridization. The concentration of the target used was 7.5 nM.
heating removes the SERRS signal due to the denaturation of the duplex, which in turn leads to the nanoparticles returning to their monodispersed form, which gives minimal SERRS. Cooling nature nanotechnology | VOL 3 | SEPTEMBER 2008 | www.nature.com/naturenanotechnology
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LETTERS reproduces the SERRS as a result of the controlled aggregation. This indicates that aggregation is critical in the control of the enhancement and that the electromagnetic contribution to enhancement is dominant. The SERRS spectra obtained from the assembled nanoparticles were investigated and it was found there is also a dependency on time, which was investigated following the addition of the complementary DNA sequence. The intensity of the signal resulting from the SERRS dye on the surface of the individual nanoparticles greatly increases over time, reaching a maximum at 60 min (Fig. 3b, inset), and can be reduced again by heating to denature the DNA and hence the aggregation state of the nanoparticles. In order to differentiate this approach from previous nanoparticle assemblies and to emphasize the control that we have over the enhancement of the Raman scattering and how beneficial this is in terms of sensitivity and detection, we prepared three batches of coded nanoparticles using different DNA sequences and dyes and specifically assembled two of the three species. The data shown in Fig. 4 indicate that the SERRS signals can be controlled by using the appropriate complementary sequence and that nanoparticles not involved in the hybridization do not need to be removed before analysis. This is the first time that coded nanoparticles have been used to produce a reproducible and clearly defined nano-assembly where the enhancement of Raman scattering from a specifically immobilized tag has switched from an effective “off ” to “on” state following the action of a biological recognition event. This is a significant result, opening up many opportunities for the combination of chemical understanding of the surface phenomenon, enhanced spectroscopy and also the biological recognition procedures. Use of different dye-coded nanoparticles with alternative probe sequences will also allow multiple sequence determination through the discriminatory power of the vibrational spectroscopy. This can be achieved through the use of differently tagged nanoparticles, in which the SERRS spectra resulting through assembly will be a mixture of the two different tags, which can be easily deconvoluted through chemometric procedures. In the first instance the most difficult stage of this is the harnessing of the enhancement phenomenon; this has been clearly demonstrated in this study. Received 25 January 2008; accepted 9 June 2008; published 11 July 2008.
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Acknowledgements The authors wish to thank the Analytical Trust Fund of the Royal Society of Chemistry for the award of the Analytical Grand Prix to D.G.
Author contributions All authors discussed the results and commented on the manuscript. D.G.T. performed all the experiments.
Author information Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/. Correspondence and requests for materials should be addressed to D.G.
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