8994 Chem. Commun., 2013, 49, 8994--8996 This journal is c The Royal Society of Chemistry 2013

Cite this: Chem. Commun.,2013,49, 8994

Enhanced Raman signals from switchablenanoparticle probes†

Phyllis F. Xu,a Ju Hun Lee,b Ke Ma,b Chulmin Choi,c Sungho Jin,a Joseph Wanga

and Jennifer N. Cha*b

We demonstrate here a DNA-based conformationally switchable nano-

dumbell structure that modulates the interparticle distance between

two gold–silver core–shell nanoparticles to induce significant changes

in the SERS signal.

Raman spectroscopy has become an increasingly important tool insensing applications because of its ability to identify specific mole-cules through their unique Raman spectra.1 To increase sensitivity,major developments have been made in Surface-Enhanced RamanSpectroscopy (SERS), where Raman signals are enhanced dramati-cally near the surfaces of noble metal films and nanoparticles.2–5

Of all the different SERS modalities, the greatest enhancementsare observed when the molecule is within a 1–2 nm gap, or a‘‘hotspot,’’ between two metal nanoparticles.6,7 Recently, systematicmethods have been developed to specifically position a Raman-activelabel within this ‘‘hotspot’’ by employing complementary oligo-nucleotides8,9 or covalent attachment of the Raman label to twometal nanoparticles.10,11 In a previous study,12 we demonstrated aswitchable nanodumbbell (NDB) structure composed of two 5 nmAuNPs where the interparticle spacings could be altered from 2 nm to7–8 nm through conformations of ssDNA hairpin or linear dsDNA. Inthis work, we have modified these initial structures to possess twoB20 nm Au@Ag core–shell NPs for increased scattering as well asincorporated a Raman-active dye site specifically within the hairpinDNA. We demonstrate first that the larger Au@Ag NPs also allowedthe NDB to switch between a linear dsDNA ‘‘open’’ state to a hairpin‘‘closed’’ state. Second, we show through both experimental andtheoretical studies that this decrease in interparticle distance withinthe NDB leads to increases in SERS enhancement that correlateclosely with the location of the dye with respect to the hotspot.

Finally, we show the potential applicability of the NDBs for biosen-sing, with ATP as a model analyte.

The NDBs consisted of two 20 nm silver coated Au nanoparticles(Au@Ag NPs) connected by a Cy3-modified dithiolated singlestranded DNA (ssDNA) (ESI†). This ssDNA was designed to form astable hairpin to bring the two Au@Ag NPs within 2 nm of eachother (Fig. 1). A portion of the dithiolated ssDNA was also designedto be complementary to an ATP aptamer (ESI†) which upon hybri-dization would cause the NDB interparticle distances to increase toB7 nm. Reaction of ATP with its aptamer would lead to dehybriza-tion12 and the dithiolated ssDNA would revert back to its hairpinstate, with the interparticle distance returning to 2 nm. (Fig. 1).

The NDB structures were first synthesized and gel extractedsimilarly to the previous work (Fig. S1, ESI†).12 Next, the AuNPs ofeach dimer were coated with silver8 to improve the SERS enhance-ment at the intended Raman laser line of 532 nm.13 The NDBs wereobtained by connecting AuNPs first and then coating with Ag becauseof the more stable conjugation between the AuNP surface and thethiolated DNA. Absorbance measurements and TEM analysis of theresulting solution (Fig. S2, ESI†) showed the Ag coating to be about5 nm thick8 with no noticeable nucleation of Ag on the dithiolatedDNA. From TEM analysis, the NDBs after Ag shell coating displayedan average interparticle spacing of 2.2� 1.0 nm (Fig. 2a and c). Thisdistance corresponds to what is expected when the NDBs are in a‘‘closed’’ hairpin formation. After the ATP aptamer was added to theAg-coated NDBs, the interparticle distances increased to an average of

Fig. 1 (a) Scheme of the NDB structural change. TEM images of (b) NDB beforeAg shell coating, (c) after Ag shell coating in hairpin formation, and (d) inextended formation, with indicated scale bars of 20 nm.

a Department of Nanoengineering and Materials Science Program, University of

California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USAb Department of Chemical and Biological Engineering, University of Colorado at

Boulder, 3415 Colorado Avenue, 596 UCB, Boulder, CO 80303, USA.

E-mail: jennifer.cha@colorado.edu; Tel: +1-303-735-6735c Department of Mechanical and Aerospace Engineering, University of

California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA

† Electronic supplementary information (ESI) available: Including DNA sequences,materials and methods, and supplementary figures. See DOI: 10.1039/c3cc44781b

Received 25th June 2013,Accepted 8th August 2013

DOI: 10.1039/c3cc44781b

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This journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 8994--8996 8995

6.9 � 2.9 nm (Fig. 2b). While the average diameter matched thetheoretical calculations, the variance can be attributed to the initialvariability in AuNP sizes, the uneven coatings of the Ag shell aroundeach particle, and possibly the flexible unhybridized ssDNA portion ofthe dithiolated strand. Addition of the ATP aptamer to these led to alarge increase in the interparticle distance, clearly showing successfulDNA hybridization, even after Ag shell formation (Fig. 2a and b).Using a more uniform AuNP or increasing the PVP concentrationduring Ag coating can alleviate the variance.

Next, SERS measurements were run to compare Raman enhance-ment between the NDBs in the ‘‘closed’’ hairpin state versus in the‘‘open’’ linear dsDNA state. When the NDBs are in their extended form,the Cy3 signals are expected to be low because the Raman dye islocated between two nanoparticles that are spaced more than 7 nm ofeach other, which is greater than the 1–2 nm distance ideal forobtaining the maximum SERS enhancement found in the closedstate.14 To compare the Raman intensities across various samples,equal numbers (between 150 and 250 NDBs, Fig. S3, ESI†) of well-dispersed NDBs on Si pillars, in either the hairpin or the linear dsDNAstate, were measured and compared (Fig. S4 and S5, ESI†). Due to thevariability in the SERS signal in single NDBs, signals from multipleNDBs were averaged to obtain an enhancement factor (EF) value.Raman signals were averaged by calculating the sum of all the Ramanintensities obtained at the most prominent fingerprint peak of Cy3 at1580 cm�1 (Fig. S6, ESI†), divided by the total number of NDBs. Bydoing so, we could obtain a value for Raman intensity per NDB,assuming that the majority of the particles were NDB dimers, whichwas verified by low magnification TEM imaging (Fig. 2c). In the originalhairpin formation, without the addition of aptamer, the EF wasdetermined to be 7.6� 104 (ESI†). When the ATP aptamer was addedto the sample, there was a significant reduction in EF to 9.6 � 103,which corresponded to almost no noticeable Cy3 peak in the actualRaman spectra (Fig. S6, ESI†). This decrease in the Raman signalshowed that the change in structural configuration from a closedhairpin to an extended state after the addition of aptamer had asignificant effect on the SERS response. The overall EF was not as largeas previously reported values,2–4 most likely due to the larger actual gapsize of the NDB, smaller size of NDB, and thinner Ag shell. However,this change in Raman intensity does match previous studies in whichlarge Raman enhancements were observed when interparticle spacingswere o2 nm but diminished rapidly as gap size increased.14

To study the effect of the position of the Cy3 molecule within thedithiolated ssDNA on SERS response, the Cy3 dye was placed atdifferent locations within the hairpin ssDNA. In the first designmentioned above (called design ‘‘E’’), the Cy3 molecule was placed

towards the 50 end of the ssDNA, right at the junction between thedouble stranded portion of the hairpin and the flexible loop domain(Fig. 3a). In the second design (‘‘HP’’), the Cy3 molecule was placeddirectly in the middle of the double stranded region of the hairpin. Inthe third design (‘‘L’’), the Cy3 molecule was placed in the middle ofthe ssDNA directly in the center of the flexible hairpin loop. Similar todesign E, the closed HP NDB showed comparable enhancementdifferences in the closed and open states (Fig. 3b), with an EF of 7.7�104 in the closed state, which decreased in the open state to 6.6� 103.Design L led to a weaker enhancement in general than either designE or HP in the closed state (EF = 4.1 � 104). This could be attributedto the fact that the Cy3 molecule in design L is in the middle of theflexible hairpin loop and therefore not well-positioned directly withinthe gap between the two Au@AgNPs.

Furthermore, the larger Raman enhancement seen in HP ascompared to either E or L is most likely due to the dye being directlywithin the double stranded portion of the ssDNA hairpin. Thispositioning led to the dye being closer in proximity to bothAu@AgNP surfaces as well as having less mobility in solution dueto the stiffness of dsDNA. The dye position did not change theinterparticle distance of the closed NDB (Fig. S7, ESI†).

To match the observed enhancement with expected enhancement,simulations were run to model the effect of NDB design on Ramanenhancement. While COMSOL simulation can only provide roughestimates of actual Raman signal enhancement, as slight variations inthe core particle size and Ag shell thicknesses and roughness havebeen shown to cause large deviations in the Raman intensity,15 wefound that the |E|4 approximation gave values for Raman enhance-ment2 that were comparable to experimental results (Fig. 3a). Asdetermined from simulation, the largest Raman enhancementoccurred for the HP NDB design (EF = 3.05 � 105) where the Cy3molecule was placed directly in the middle of the hybridized domainof the hairpin, followed by the E NDB system (EF = 1.87 � 105).

Fig. 2 TEM histogram analysis of interparticle distances. (a) NDBs in the naturalhairpin form, (b) and after addition of aptamer in the extended form. (c) TEMimage showing majority dimers after gel extraction.

Fig. 3 (a) COMSOL simulations of NDB structures with ‘‘open’’ and ‘‘closed’’structures for various positions of Cy3 dye. Experimental and theoretical approx-imations of the Cy3 position within the NDB gap are shown. The positioning ofthe Cy3 is shown as the red star. (b) Comparison of average intensity/NDB of NDBdesigns in ‘‘open’’ and ‘‘closed’’ states. Intensity is taken at the Cy3 fingerprintpeak at 1580 cm�1.

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8996 Chem. Commun., 2013, 49, 8994--8996 This journal is c The Royal Society of Chemistry 2013

The smallest Raman signal was seen for design L (EF = 1.7 � 104),where the Cy3 was in the flexible hairpin region and thus fartheraway from the interparticle gap. The theoretical EF estimates weredetermined by using Cy3 positions within the gap that werecalculated based on the lengths of the ssDNA and dsDNA. As acomparison, the actual positions of the Cy3 dye within the open andclosed NDBs were also determined using the experimentally derivedEF values from Raman measurements. As shown in Fig. 3a, theslightly smaller EF values of the experimentally measured NDBsdemonstrated that the actual location of the dye molecule withinthe interparticle gap of each NDB was slightly different from thatexpected from DNA length calculations. One reason for this dis-crepancy may be that the flexible T15 sequences located at the 50 and30 ends of the dithiol ssDNA were not completely covered by the Agshell and therefore led to slight variations in dye location.

In future work this may be fixed by growing the Ag shell slightlythicker to limit the number of unhybridized thymines at the 50 and 30

ends of the closed hairpin. Despite these small discrepancies, how-ever, all of the EF values obtained through simulation for the openand closed NDB states were within one order of magnitude of theexperimentally obtained values. Therefore, applying the |E|4 approxi-mation in the COMSOL simulations appears viable in this instance.

Since the dithiolated ssDNA hairpin DNA within the NDBs werepartially complementary to an ATP aptamer, competitive binding byATP with the complementary aptamer strand could cause the NDBs toswitch from linear to hairpin, leading to a large increase in the Ramansignal. To test for this, ATP was introduced into a solution of NDBswith the E design. As shown in Fig. 4a, the addition of 1 mM ATP to a330 pM solution of open NDBs (design E) caused the competitiveremoval of the aptamer strand, allowing the NDB structure to revertback to the closed state with the reformation of the hairpin. By doingso, the interparticle distance decreased to 3.1 � 1.3 nm (Fig. 4a),which is slightly larger than in the as-made NDB closed state. Thiscould be due to annealing of the as-made NDB compared to constanttemperature incubation of the NDB after ATP addition, resultingin possibly incomplete hairpins and larger interparticle distance.

The addition of ATP also led to a 7-fold gain in the Raman signaland the resulting intensity was similar to the original intensity of theNDB structure in the hairpin state prior to addition of the aptamerstrand (Fig. 4b and c). While a significant increase in Raman intensityafter the addition of ATP is necessary for sensing, it is just asimportant to have very low background signals from the inactivatedNDBs. Because the NDBs in the open state showed consistently lowRaman intensities, it was possible to detect small increases in theRaman signal, enabling the possibility of using these NDB structuresfor ‘‘on/off’’ sensing applications. Quantitative values of ATP may beobtained, but it should be noted that absolute values could be off dueto existing standard deviations in average intensity/NDB (Fig. S8,ESI†). In contrast to fluorescence-based molecular beacons wheresmall fluctuations in distance can lead to large variations in thebackground signal15 the low-noise levels observed with these NDBsmay allow higher sensitivities and signal-to-noise ratios.

We have demonstrated a nanodumbbell structure that exhibitslarge changes in surface-enhanced Raman signal through altera-tions in structural conformation. When the NDBs were configuredto be in the open state by using linear dsDNA, the large interparticlegap between the Au@Ag NPs led to low Raman signals from theCy3. After switching to an ssDNA hairpin state, the decrease ininterparticle distance to B2 nm caused a significant increase inRaman intensities. Through experimental and theoretical studies,we measured Raman enhancement for three different NDB designsand we showed that having the Cy3 placed directly within thedouble stranded portion of the hairpin led to the largest Ramanenhancements. Furthermore, by using an aptamer sequence forATP as a complement to the hairpin strand, we showed that theseNDBs could potentially be used for sensing applications.

The authors thank Dr Andrew Goodwin for manuscript editingand Professor Frank Talke for the use of his Raman microscope. PFXis financially supported by the Department of Energy (DE-SC0004937)and the Office of Naval Research (Award Number: N00014-09-01-0250). JHL is supported by NSF CAREER (DMR-1056808). JW is alsosupported by the National Science Foundation Award CBET-1066531.

Notes and references1 B. Singh, R. Gautam, S. Kumar, B. N. Vinay Kumar, U. Nongthomba,

D. Nandi, G. Mukherjee, V. Santosh, K. Somasundaram andS. Umapathy, Curr. Sci., 2012, 102, 232.

2 K. Kneipp, H. Kneipp, I. Itzkan, R. R. Dasari and M. S. Feld, J. Phys.:Condens. Matter, 2002, 14, R597.

3 G. Haran, Acc. Chem. Res., 2010, 43, 1135.4 K. Kneipp, Y. Wang, H. Kneipp, L. T. Perelman, I. Itzkan,

R. R. Dasari and M. S. Feld, Phys. Rev. Lett., 1997, 78, 1667.5 S. E. J. Bell and N. M. S. Sirimuthu, J. Am. Chem. Soc., 2006, 128, 15580.6 J. P. Camden, J. A. Dieringer, Y. Wang, D. J. Masiello, L. D. Marks, G. C.

Schatz and R. P. Van Duyne, J. Am. Chem. Soc., 2008, 130, 12616.7 E. C. Le Ru, P. G. Etchegoin and M. Meyer, J. Chem. Phys., 2006,

125, 204701.8 D. Lim, K. Jeon, H. M. Kim, J. Nam and Y. D. Suh, Nat. Mater., 2010, 9, 60.9 N. H. Kim, S. J. Lee and M. Moskovits, Adv. Mater., 2011, 23, 4152.

10 N. Guarrotxena and G. C. Bazan, Chem. Commun., 2011, 47, 8784.11 N. Guarrotxena, B. Liu, L. Fabris and G. C. Bazan, Adv. Mater., 2010,

22, 4954.12 P. F. Xu, A. M. Hung, H. Noh and J. N. Cha, Small, 2013, 9, 228.13 R. J. Stokes, A. Macaskill, P. J. Lundahl, W. E. Smith, K. Faulds and

D. Graham, Small, 2007, 3, 1593.14 J. Lee, J. Nam, K. Jeon, K. Lim, H. Kim, S. Kwon, H. Lee and Y. D.

Suh, ACS Nano, 2012, 6, 9574.15 N. Hamaguchi, A. Ellington and M. Stanton, Anal. Biochem., 2001,

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Fig. 4 (a) Histogram of interparticle distance of NDB after ATP addition withcorresponding TEM image. (b) Comparison of average Raman intensity per NDBand (c) sample SERS spectra of original NDB structures, after aptamer addition,and after ATP addition.

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