Shell-Engineered Chiroplasmonic Assemblies of Nanoparticles for

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Shell-Engineered Chiroplasmonic Assemblies of Nanoparticles for Zeptomolar DNA Detection Yuan Zhao,† Liguang Xu,† Wei Ma,† Libing Wang,† Hua Kuang,*,† Chuanlai Xu,† and Nicholas A. Kotov*,‡,§,∥,⊥ †

State Key Lab of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, P. R. China ‡ Department of Chemical Engineering, §Department of Materials Science, ∥Department of Biomedical Engineering, and ⊥ Biointerfaces Institute, University of Michigan, Ann Arbor, Michigan 48109, United States S Supporting Information *

ABSTRACT: DNA-bridged pairs of seemingly spherical metallic nanoparticles (NPs) have chiral geometry due to the nonideal oblong shape of the particles and scissor-like conformation. Here we demonstrate that deposition of gold and silver shells around the NP heterodimers enables spectral modulation of their chiroplasmonic bands in 400− 600 nm region and results in significantly enhanced optical activity with g-factors reaching 1.21 × 10−2. The multimetal heterodimers optimized for coupling with the spin angular momentum of incident photons enable polymerase chain reaction (PCR)-based DNA detection at the zeptomolar level. This significant improvement in the sensitivity of detection is attributed to improvement of base pairing in the presence of NPs, low background for chiroplasmonic detection protocol, and enhancement of photon− plasmon coupling for light with helicity matching that of the twisted geometry of the heterodimers. KEYWORDS: Nanoparticle assemblies, chiroplasmonic properties, core−shell deposition, amplification, biosensing

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hiral plasmonic assemblies,1−4 such as gold (Au) helix bundles,5−7 pyramids,8,9 chains,10−12 dimers,13−15 and other superstructures,16,17 display a uniquely strong polarization rotation that makes them a remarkable new class of chiroptical materials. Circular dichroism (CD) spectroscopy is not a typical analytical tool because the difference in absorption of left- and right-circularly polarized light (CPL) is small in most molecules. However, strong coupling between spin angular momenta of incoming photons and plasmons/excitons in chiral nanostructures may change this perception, and chiral plasmonic assemblies display significant potential for biosensing.18−20 Identification of DNA is vital for disease diagnostics, forensics, and environmental science. Zeptomolar DNA detection is critical for the early detection of cancer, gene mutations, and infection. Optimizing the sizes and shapes of building blocks of chiral nanoscale assemblies can potentially make it possible to enhance the intensity of CD peaks with small amplitude, but obtaining tunable and strong chiroptical signals is a significant challenge.15,21 Another less explored mechanism for CD signal amplification and reversible regulation may be realized by deposition of additional layers of Au or Ag on preassemblies to create the core−shell geometry of constitutive nanoparticles (NPs). In the past, the deposition of metallic shells around NPs resulted in substantial improvement of limits of detection (LODs) for colorimetric, surfaceenhanced Raman scattering, and electrochemical processes.22−28 © XXXX American Chemical Society

NP dimers represent DNA-bridged terminal assemblies with intrinsic self-limitation in the size and number of assembled building blocks due to the singularity of DNA ligands attached to their surface. Due to this property they can be synthesized with high homogeneity and precision. NP dimers were recently identified to be chiral structures despite their seemingly axial and/or centrosymmetric geometry.15,20 This chiroptical activity of NP dimers originates from their oblong geometry combined with a slight twist between long axes of the particles. The resulting difference in absorption of left and right CPL is large due to the strong coupling of the spin angular momenta of the incident photons with plasmonic assemblies. Here, Au heterodimers (HDs) were assembled through a polymerase chain reaction (PCR) taking advantage of exponential amplification of target oligonucleotides, enhanced specificity of the method, and an improved base-paring process in the presence of metal NPs.3,12,16,29 We investigated the chiroplasmonic activities (intensity and peak position) of HDs after depositing gold or silver shells around them. It was found that CD bands of HDs can be tuned from 418 to 586 nm by varying shell thickness, composition, and the sequence of the metallic layer. The g-factor of core−shell HDs was found to be as high as 1.21 × 10−2, enabling zeptomolar DNA detection of long Received: March 30, 2014 Revised: May 8, 2014

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Figure 1. Schematic illustration of (a) PCR-assembled HDs and (b) sequential post-assembly deposition of Ag and Au shell(s).

Figure 2. Representative TEM images of HDs after (a) 2, (b) 5, (c) 10, and (d) 20 PCR cycles (insert, 0th cycle). (e) Statistical analysis of TEM images with respect to different assembly products. (f) CD and UV−vis spectra of the NP assemblies obtained for 0, 2, 5, 10, and 20 PCR cycles.

those observed for Au nanorods.14,20,30 Also, Coulombic interactions between chiral molecules and plasmonic modes may contribute to the polarization rotation of photons in the visible spectral interval.20,31 The geometry of Au NPs was confirmed to be ellipsoidal by cryo-electron tomography (Figure S6). The aspect ratios of Au NPs in this study were found to be 1.15 ± 0.08 and 1.10 ± 0.05 for 25 ± 3 nm and 10 ± 2 nm NPs, respectively. The angle between the long axes of the NPs was ∼10° (Figures S1, S6−S7). Therefore, the observed CD bands originate from the interactions of the circularly polarized photons with twisted NP pairs of the same helicity. Note that the shoulder(s) in the 400−500 nm range should be attributed to “dark” transitions of coupled plasmons32,33 that acquire some oscillator strength in the twisted configuration. The assignment of the experimental CD spectra to twisted pairs of elongated NPs was also confirmed by simulations using the frequency domain of the finite integral method (Figure S17, black curve). The intensity of the CD band at 525 nm increased by about a multiple of four as the number of PCR cycles increased from 2 to 20. The positions of the peaks in the UV−vis spectra were

strands of DNA that has until now been difficult by traditional and NP-based methods (Tables S1 and S2). PCR was performed on the surface of 25 ± 3 nm and 10 ± 2 nm Au NPs carrying one forward (F50) and reverse primer (R50) respectively (Figure 1 and Figure S1). The selectivity of the modification of the NP with the primers was achieved by judicious optimization of the synthesis and separation conditions described in the Methods and SI. The amplification length of primers was 50 base pairs (bp). The PCR step increased the yield of HDs, simplified, and automated their assembly process. The yields of HDs increased gradually from 35.6% ± 1.2% for two cycles to 78.9% ± 2.4% for 20 cycles with optimized PCR amplifying procedures (Figure 2a−e and Figures S2−S5). Individual NPs showed smaller hydrodynamic diameters than those of HDs, which matched our expectations (Figure S1). CD spectra of HDs revealed bands at 260 and 525 nm. The former is attributed to electronic excitations typical for the chiral secondary structures of dsDNA (Figure 2f) altered by interactions with NPs. A CD band at 525 nm is attributed to the intrinsic chiral geometry of the pairs of elliptical Au NPs having positive angles between the main axes of NPs similar to B

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Figure 3. HDs carrying (a, b) Ag and (c, d) Au shells with increasing thickness. (a) Representative TEM images of core−shell HDs made by addition of 30, 50, 70, and 100 μL of solution of 1 mM AgNO3 (from left to right then up to down). (b) CD and UV−vis spectra of HDs with Ag shell. Insert, photographs of corresponding HD dispersions. (c) Representative TEM images of core−shell HDs made by addition of 10, 20, 30, and 50 μL solution of 5 mM HAuCl4. Scale bars 20 nm. (d) CD and UV−vis spectra of HDs with the Au shell. Insert, photographs of different HD dispersions. Sample 1: original HDs. Samples 2−8: core−shell HDs made by addition of 5, 10, 20, 30, 50, 70, and 100 μL solution of 1 mM AgNO3. Samples 9−13: core−shell HDs made by addition of 5, 10, 20, 30, and 50 μL solution of 5 mM HAuCl4.

g-factors reported for most organic and biological compounds.5 We attribute such high ellipticity to two structural features of the coated HDs. First, Ag shell deposition reduced and eventually bridged the interparticle gaps of HDs. Hence, the hybridization of the plasmonic dipoles became stronger.5 Second, the deposition of Ag shell also increased the aspect ratio of NPs in the assemblies and was completely unexpected. As such, after deposition of a 9.9 ± 1.5 nm Ag shell (100 μL of AgNO3 solution), the aspect ratio of NPs with 25 nm Au cores increased from 1.15 ± 0.08 to 1.31 ± 0.09. The same was also true for Au NPs with 10 nm cores, whose aspect ratio increased from 1.10 ± 0.05 to 1.28 ± 0.07 after deposition of a 6.1 ± 1.2 nm Ag shell (100 μL AgNO3 solution) (Figure S7). The increase in aspect ratio of NPs increases the photonic interaction of the twisted NP assemblies of the same helicity as the incoming photons, resulting in the enhancement of the CD band intensity (Figures 3b and S11).2,30 Also note that encapsulation of DNA strands in metal also makes a contribution to optical activity of the HDs with shells (Figure 3b).5 We also explored how deposition of Au shell affects optical properties of HDs. HDs@Au were made by addition of different amounts (5−50 μL) of 5 mM solution of HAuCl4 to HDs. Note that the surface of HD@Au was more corrugated than for HD@Ag (Figure 3c, Figures S13−S14). This difference was attributed to the attachment of small gold NPs serving as intermediates in the process of shell formation; they promote “spiky” morphologies of the resulting NPs.37 As the average thickness of the Au shell increased, the color of the dispersion changed from pink to purple (Figure 3d). Compared to the original HDs, the chiroplasmonic band at 525 nm in CD spectra of HD@Au exhibited a 61 nm red-shift (Figure 3d), similar to red-shifts of UV-bands in tight NP agglomerates (Table S1). Similarly to the effect of the Ag shell, the addition

similar to those originally observed for NPs (Figure 2f) because the electromagnetic coupling between plasmonic NPs connected by 50 bp DNA strands is weak.24,30,34 This is typical for all NP assemblies connected by long DNA strands (Table S1).24,35,36 When the number of PCR cycles was increased from 25 to 40, higher oligomers appeared in addition to HDs. The loss of regularity in the sign of dihedral angles between the long axes of NPs in HD oligomers led to decreased chiroptical activity (Figure S8).29 Overall, HD dispersions obtained after 20 cycles showed the highest polarization rotation and were selected for further studies. A single shell of Ag or Au (denoted as @Ag and @Au, respectively) was deposited on HDs to further increase their chiroplasmonic activity. Ag shells with different thicknesses were formed by adding different amounts (5−100 μL) of a 1 mM solution of AgNO3 (Figure 3a−b). In concert with the increased amount of Ag+ ions, we observed (a) a thickening Ag layer in TEM images, (b) an increase of Ag content from 5.0% ± 0.8% to 76.5% ± 1.3% in EDX data (Figure S9), and (c) HD hydrodynamic diameters by dynamic light scattering (DLS) (Figure S10). The color of HD dispersions changed from the original pink to yellow, indicating the dominance of Ag plasmons in HD@Ag (Figure 3b). As the thickness of the Ag shell increased, the CD bands increased in intensity (Figure S11) and shifted from 525 to 418 nm (Figure 3b), which demonstrated the possibility of “tuning” both the intensity and the spectral position of the chiroplasmonic response of NP assemblies.28 The match in spectral position and shape of the calculated and experimental CD and UV−vis bands (Figure S17) confirmed the attribution of the optical properties of the core−shell structure of HD@Ag. The dimensionless parameter describing optical anisotropy of chiral assemblies, i.e., g-factor (see SI), reached 1.03 × 10−2 for HD@Ag (Figure 3a−b, Figure S12a), which is higher than C

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Figure 4. (a) Scanning TEM-EDX elemental map and CD and UV−vis spectra of double shell HDs with the first Ag shell and then the Au shell. Samples 1−5: double shell HDs made by addition of 5, 30, 50, 70, and 100 μL of solution of 1 mM AgNO3 and then 0.8, 3, 5, 6, and 8 μL of solution of 5 mM HAuCl4. (b) Scanning TEM-EDX elemental map and CD and UV−vis spectra of double shell HDs with the first Au shell and then the Ag shell. Samples 6−10, double shell HDs made by addition of 5, 10, 20, 30, and 50 μL solution of 5 mM HAuCl4 and then 5, 10, 30, 40, and 50 μL solution of 1 mM AgNO3. Scale bars for (a) and (b) are 20 nm.

(Figure 5). The linear range of the analyte detection was found to be from 160 zM to 1.6 pM; it spans 7 orders of magnitude of

of the Au shell amplified the intensity of the CD band. The strongest CD band for HD@Au was at 586 nm with a g-factor of 1.21 × 10−2, which was higher than that of HD@Ag (Figure S12b). The corrugated conformation of the HD@Au surface and the decreasing gaps between two NPs after Au shell deposition resulted in enhancement of the CD bands. Deposition of double shells on HDs can be also exploited to expand the palette of CD bands suitable (Figures S15 and S16). The double shells can be made by the sequential deposition of Ag shell first and then Au shell later or vice versa following the same general processes as above. For instance, the addition of 30 μL of a 1 mM AgNO3 solution to the dispersion of DNAbridged HDs was followed by addition of 3 μL of 5 mM HAuCl4 solution. The formation of double shells around both NPs in HDs was confirmed by energy dispersive X-ray (EDX) elemental mapping (Figure 4). The superimposed elemental maps revealed that Au was located in the core of the particles; the sequence of the surrounding shells agrees with the sequence of exposure to AgNO3/HAuCl4 solutions. Not entirely surprisingly, after observing blue- and red-shifts of the principle CD peaks, chiroptical bands for HD@Ag@Au and HD@Au@ Ag returned to ca. 525 nm, as observed previously for HDs. Multimetal shells provide a new way to “tune” the peak position of CD bands; the experimental results agreed with predictions made by finite integral method simulations (Figure 4 and Figure S17). The g-factors for HD@Ag@Au and HD@Au@Ag were found to be 0.44 × 10−2 and 0.28 × 10−2 (Figure S12c− d), respectively. Strong CD bands of DNA-bridged HDs enable utilization of this technique in DNA detection. HD@Au not only displayed fairly narrow CD spectra but also possessed the highest g-factor (1.21 × 10−2) in this study. Therefore, these NP assemblies were deliberately selected for the study of their prospects in biosensing. The specific design of primers is critical for high selectivity in DNA detection, and interface PCR-based chiroplasmonic methods further improved detection specificity owing to local heat transport in the media.3 DNA concentration determines the amount of HD@Au and hence has direct connotations to the optical activity of the resulting dispersions. A calibration curve for chiroplasmonic DNA detection with core−shell HDs was obtained by the standard dilution method

Figure 5. (a) CD and UV−vis spectra of HDs made by addition of 50 μL of solution of 5 mM HAuCl4 for DNA concentrations from 16 zM to 1.6 pM. (b) Analytical calibration curve relating the intensity of CD bands of HD@Au and the concentration of DNA.

DNA concentrations (Figure 5). The LOD was as low as 17 zM; it can be compared to the LOD of 500 zM for colorimetric biobar-code assay27 and the LOD of 1 aM for fluorometric quantum-dot based detection (Table S2).38 Fluorescence quantitative-PCR (FQ-PCR) is performed at the same standard DNA concentrations, and the results validate the effective calibration process of the chiroplasmonic method (Figure S18). In comparison to methods using fluorescence labels, the chiroplasmonic method circumvents some sources of variability related to the use of photolabile fluorophores and uses the CD signal in a part of the spectrum that has contribution and therefore noise from other components. The calibration curve D

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with respect to DNA concentration obtained for five independent test samples confirms the high accuracy of chiroplasmonic method (Figure S19, Table S3). The factors resulting in the amplified polarization rotation in HDs that enable such low detection limits are multiple: (a) the angled geometry of plasmonic NP HDs with high S/N ratio due to low background at plasmonic frequencies; (b) coating with additional layers of metal silver with high plasmon intensity enhances the intensity of the signal; (c) improvement of the base-paring process in the presence of NPs due to improved uniformity of the temperature in the media;3 (d) and strong coupling of the incoming photons with the plasmonic systems despite the wide gap separating the particles. The latter property distinguishes the chiroplasmonic method from all other techniques39 using metal NPs because it does not require the formation of “hot-spots” and significant electrical field enhancement in the gap.10 This feature contributes to its selectivity since longer DNA strands have better target specificity. In conclusion, chiral assemblies were nanoscale-engineered to enhance their chiroptical activity by deposition of additional metallic shells. Different shell types, thicknesses, and layer sequences makes it possible to tailor their chiroptical activity with respect to spectral position and intensity. Spectral tunability of the CD bands is most essential for multiplexing of DNA analysis that often requires identification of several genes simultaneously. The amplified chiroptical signal combined with the exponential amplification of PCR enables core−shell assemblies to achieve zeptomolar DNA detection. Further improvements of the chiroplasmonic method aimed at even stronger coupling with the rotational component of visible photons are possible by optimizing the size and geometry of the NP assemblies.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental methods, details of model calculation, and accompanying figures and tables. This material is avaiable free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions

Y.Z., L.X., and W.M. contributed equally to this paper. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is financially supported by the Key Programs from MOST (2012BAC01B07), NSFC (21101079, 21371081, 21301073), and grants from MOE (NCET-12-0879). This material is based upon work partially supported by the Center for Solar and Thermal Energy Conversion, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Award Number no. DE-SC0000957, and the ARO MURI W911NF12-1-0407 “Coherent Effects in Hybrid Nanostructures for Lineshape Engineering of Electromagnetic Media” (N.A.K.). We acknowledge support from NSF under grant CBET 1036672 (N.A.K.). E

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