Plasmon Enhanced Fluorescence in Coupled Nanostructures and

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Plasmon Enhanced Fluorescence in Coupled Nanostructures and Applications in DNA Detection Zhijun Zhu, Peiyan Yuan, Shuang Li, Monalisa Garai, Minghui Hong, and Qing-Hua Xu ACS Appl. Bio Mater., Just Accepted Manuscript • Publication Date (Web): 26 Apr 2018 Downloaded from http://pubs.acs.org on May 22, 2018

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ACS Applied Bio Materials

Plasmon Enhanced Fluorescence in Coupled Nanostructures and Applications in DNA Detection Zhijun Zhu,1,2 Peiyan Yuan,1 Shuang Li,1 Monalisa Garai,1 Minghui Hong,3 and QingHua Xu1* 1

Department of Chemistry, National University of Singapore, 3 Science Drive 3,

Singapore 117543 2

Department of Materials Science and Engineering, Qingdao University, 308 Ningxia

Road, Qingdao, China 266071 3

Department of Electric and Computer Engineering, National University of

Singapore, 4 Engineering Drive 3, Singapore 117583

Abstract: Plasmon coupling interactions between adjacent noble metal nanoparticles (NPs) can cause significantly enhanced local electric field in the gap region, which could be utilized to dramatically enhance the fluorescence intensity of chromophores. Here we performed a systematic study on the influence of different factors on Plasmon coupling enhanced fluorescence, including shape and size of metal NPs, dye distribution and separation distance. Cyanine 5 (Cy5) acted as the fluorescence probe and DNA was employed to assemble nanostructures to immobilize Cy5 into the gap region of the coupled metal NPs. Fluorescence of Cy5 was pre-quenched by attaching DNA linked Cy5 to the surface of Au nanospheres (NSs). The quenched fluorescence of Cy5 was turned-on by forming nano-assembly through DNA hybridization with different enhancing substrates: Au NSs, Au nanorods (NRs) and Au@Ag NSs. Au@Ag NSs were found to give the largest fluorescence enhancement effect. Larger sized Au@Ag NPs were found to display larger fluorescence enhancement effects compared to the smaller ones. Optimum fluorescence enhancement was observed at an intermediate inter-particle separation distance of 8.2 nm, which was up to 100-fold 1 ACS Paragon Plus Environment

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enhancement compared to quenched Cy5-Au NSs, 5-fold enhancement compared to unquenched free Cy5 molecules. As pre-quenched fluorescence offers reduced background, this Plasmon coupling enhanced fluorescence phenomenon was further utilized to develop a simple fluorescence turn-on platform for highly sensitive and selective detection of DNA sequence. The limit of detection (LOD) of this method was estimated to 3.1 pM. The exceptional selectivity of this method allows to distinguish single-base mismatch at the room temperature. This Plasmon coupling enhanced fluorescence phenomenon could be further utilized to develop various platforms for highly sensitive sensing and imaging applications.

Keywords: Metal enhanced fluorescence, Plasmon coupling, Noble metal nanoparticles, Plasmon resonance, Bio-sensing, DNA detection

Introduction Noble metal nanoparticles (NPs) have attracted much attention due to their unique optical properties and wide applications in various fields.1 In particular, they display an interesting property known as localized surface Plasmon resonance (SPR),2 which arises from collective oscillation of conduction band electrons upon interaction with light. SPR of noble metal NPs depends on the composition, morphology of metal NPs , the surrounding dielectric environment as well as coupling interactions between adjacent metal NPs. SPR of metal NPs creates a strongly localized electric field near the particle surface, which could significantly modulate the optical properties of nearby chromophores. Various metal enhanced optical responses have been reported, such as surface-enhanced Raman scattering (SERS), metal enhanced fluorescence3 and various nonlinear optical responses.4-6 2 ACS Paragon Plus Environment

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Metal-enhanced fluorescence has emerged as an exciting new research area, which has attracted lots of interest in both fundamental studies and practical applications.3,7 Fluorescence intensity of chromophores could become significantly modulated upon interactions with noble metal NPs. Plasmonic interactions between metal NPs in the coupled nanostructures have been known to cause significantly enhanced electric fields within the gap region, which has been utilized to obtain giant SERS signals.7 Plasmon coupling induced local electric field amplification could also result in enhanced excitation efficiency and increased radiative decay rates of the chromophores located at the gap region, therefore significantly enhancing their overall fluorescence intensities. Coupled metal NPs have been shown to display stronger fluorescence enhancement effects compared to uncoupled metal NPs.8 Similar to enhanced fluorescence by individual metal NPs, plasmon coupling enhanced fluorescence is also expected to depend on the morphology and composition of metal NPs, quantum yield of fluorophores, the metal-fluorophore separation distance, as well as spectral overlap between SPR of metal NPs and absorption/emission spectra of the fluorophores.9-11 In addition, it is also expected to depend on the coupling strength that is determined by separation distance between coupled metal NPs. It is critical to design coupled nanostructures with proper plasmonic properties to achieve large fluorescence enhancement. Using various assembly methods, plasmon coupling enhanced fluorescence has been previously achieved in the studies of both ensemble samples and on the single molecule level.1215

Using a bowtie nanoantenna structure, Kinkhabwala et al. demonstrated 3 ACS Paragon Plus Environment


enhancement of 1340-fold in emission intensity of TPQDI.16 The fluorescence intensity Atto-665 was enhanced by 170-fold by the aggregated Ag NPs17 and 470fold by a tip-to-tip Au NR dimer.18 Using three-dimensional plasmonic nano-antennadots array, Zhou et al. demonstrated giant enhancement of 7400-fold in fluorescence intensity of IgG.19 DNA has been known as an excellent platform to form assembled nanostructures due to their precise control, desired modification, and specific recognition.20 In addition, DNA can be easily functionalized with various fluorophores.21-23 DNA could therefore be employed to assemble nanostructures to immobilize fluorophores within the hotspot location of the coupled NPs.24 Herein, we present a systematic study on different factors that might influence Plasmon enhanced fluorescence in the coupled metal nanostructures, including shape and size of metal NPs, dye distribution and separation distance. Cyanine 5 (Cy5) acted as the fluorescence probe. Upon attaching DNA linked Cy5 to the surface of Au NSs, the fluorescence of Cy5 was quenched through energy transfer to Au NPs to obtain an “off” state. The quenched fluorescence of Cy5 was turned-on upon coupling interactions with various metal NPs. The optimum nanostructure that give largest fluorescence enhancement was screened through systematical studies. The optimum fluorescence enhancement was obtained by using Au@Ag core-shell NPs, which gave 100-fold enhancement compared to quenched Cy5-Au NSs, 5-fold enhancement compared to unquenched free Cy5 molecules. As pre-quenched fluorescence offers reduced background, large Plasmon induced fluorescence enhancement renders large 4 ACS Paragon Plus Environment

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contrast for highly sensitive detection. This Plasmon coupling enhanced fluorescence phenomenon has been further utilized to design a simple turn-on platform for highly sensitive and selective detection of DNA sequence. Single-base mismatch can be easily distinguished even at the room temperature. Table 1. Oligonucleotides used in this work. Name

Sequence (from 5’ to 3’)

oligo0

T10-(CH2)3-SH

oligo1

Cy5-T10-(CH2)3-SH

oligo2

GTC CGT CTT GTC T10-(CH2)3-SH

oligo3

HS-(CH2)6-T10 CTG TGC TTC CTG

oligo4

GAC AAG ACG GAC T10-(CH2)3-SH

oligo5

Cy5-GTC CGT CTT GTC T10-(CH2)3-SH

target24

GAC AAG ACG GAC CAG GAA GCA CAG

target36

GAC AAG ACG GAC AAG TGA ATC TAG CAG GAA GCA CAG

target36’

CTA GAT TCA CTT

1-mismatched target

GAC AAG ACA GAC CAG GAA GCA CAG

2-mismatched target

GAC AAG ACA GAC CAG GAC GCA CAG

3-mismatched target

GAC AAG ACA GAC CTG GAA GCA TAG

Experimental Chemicals: Phosphate buffered saline buffer (PBS, 10 mM PB, 0.137 M NaCl, pH 7.4) was purchased from Invitrogen. All the oligonucleotides (Table 1) and other chemicals were obtained from Sigma. All reagents were used as received without further purification. Milli-Q water (18.2 MΩ·cm) was used in all the aqueous solution in the experiments. Instruments: Absorption and fluorescence spectra of various solutions were 5 ACS Paragon Plus Environment


measured on a UV-vis spectrophotometer (Shimadzu, Japan) and a FluoroMax-4 fluorometer (Horiba, USA), respectively. Transmission electron microscopy (TEM) images were taken on a JEOL 2010 microscope at an accelerating voltage of 200 kV. Fluorescence lifetimes were measured by using the time-correlated single photon counting (TCSPC) method (Picoquant GmbH, Berlin, Germany) with second harmonic generation (410 nm) of the output of a femtosecond Ti:sapphire oscillator (Avesta TiF-100 M) as the excitation source and a photomultiplier tubes (PMT, PicoQuant) as the detector, which gives a temporal resolution of ~100 ps. Preparation of metal NPs: Au NSs of different sizes were prepared by reduction of HAuCl4 with various amounts of sodium citrate in the aqueous solution.25 To prepare Au@Ag NSs, 17 nm Au NSs were first prepared as the seed. 3.0 mL of the asprepared 17 nm Au NSs seed solution was added into 27 mL of aqueous mixture solution containing 4.0 mM sodium citrate and 1.0 mM ascorbic acid. After adjusting the pH of solution to ~7 using NaOH, 1.0 mL of 18 mM AgNO3 aqueous solution was added dropwise under stirring for 30 min. The solution was then transferred into an oil bath and refluxed for another 1 h. The thickness of Ag shell was controlled by using different amount of AgNO3 solution. The obtained nanoparticle solutions were stored at 4°C before its use. Preparation of oligonucleotide modified Au NSs, Au NRs and Au@Ag NSs: DNA modified Au NSs were prepared according to a previous report.26 The disulfide group on the oligonucleotides was first cleaved by using DTT. The resultant mixtures were incubated at room temperature for 1 h in the dark and then purified with a NAP-5 6 ACS Paragon Plus Environment

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column. 50 µL of 20 µM freshly cleaved DNA was added into 500 µL of 2 nM Au NSs. T10-(CH2)3-SH (Oligo0, see Table 1) was utilized to adjust the amount of Cy5DNA and hybridizing DNA. The mixture was incubated at room temperature overnight. After adding NaCl to reach the concentration of 30 mM, the mixture was sonicated for 10 s. After incubation the salting process was repeated every two hours at room temperature to reach a final NaCl concentration of 0.5 M. The unbound DNA was removed by repeated centrifugation and re-dispersion in water. The obtained solution was then stored at 4 °C before its further use. DNA modified Au@Ag NSs were prepared by using the same procedure. DNA modified Au NRs were prepared according to a previous report.27 Fluorescence quenching of Cy5 by Au NSs Generally, fluorescence of Cy5 will be quenched upon attaching to the surface of Au NSs. To accurately determine the extent of fluorescence quenching, fluorescence spectra of Cy5-DNA functionalized Au NSs were measured before (quenched) and after (unquenched) incubation with 0.1 M DTT for overnight at room temperature to release Cy5-DNA from the surface of Au NSs into the solution.26 Assembly of the nanostructures and fluorescence measurements: Desired nanostructures were assembled through hybridization of surface modified DNA strands with specific sequences. Desired concentration of the complementary DNA was added into the mixtures of Cy5-DNA modified Au NSs (0.4 nM) and DNA modified enhancing substrates (Au NSs, Au NRs or Au@Ag NSs). The as-prepared mixture was kept in a water bath at 70 °C for 5 min and then slowly cooled down to 7 ACS Paragon Plus Environment


room temperature before the measurements of fluorescence spectra under excitation at 600 nm. All fluorescence spectra were corrected by subtracting the background contribution from the nanoparticles. Separation distance-dependent fluorescence enhancement measurements: The gap distance (4.1, 8.2 and 12.2 nm) between Au NSs and the enhancing substrates (Au@Ag NSs) were controlled by using ds-DNA with different lengths. 4.1 nm gap distance were obtained by hybridization of oligo5 modified Au NSs with oligo4 modified Ag NSs; 8.2 nm gap distance was obtained by hybridization of oligo5 with target24; 12.2 nm gap distance was obtained by hybridization of oligo5 with target36 and target36’. The mixture solutions were kept overnight in PBS to ensure full hybridization. The samples were subsequently washed three times to remove excess DNA and then dispersed in PBS for further use. The coupled nanostructures with different gap distances were finally obtained by hybridization of these three ssDNAAu NSs samples and oligo3 labeled Au@Ag NSs respectively. DNA detection assay: A series of 480 µL of oligo5 modified Au NSs (0.4 nM) and oligo3 labeled Au@Ag NSs (0.4 nM) in PBS solution were mixed with 20 µL of target24 stock solutions to give the desired concentrations. The mixture solution were first heated to 70 ºC and then slowly cooled down to room temperature to allow hybridization of DNA strands before the fluorescence spectra were measured.

Results and discussion The overall design of the assay is shown in Scheme 1. The basic principle is based on 8 ACS Paragon Plus Environment

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fluorescence quenching and enhancement effects of plasmonic metal nanostructures.4 The fluorescence of Cy5 will be generally quenched upon attaching to the surface of Au NSs,28 resulting in an “off” state. The quenched fluorescence of Cy5-Au NSs could be turned on upon coupling interactions with the enhancing substrates. Assembly of Cy5-Au NSs and different ssDNA modified enhancing substrates (Au NSs, Au NRs and Au@Ag NSs) were achieved by formation of double stranded DNA duplexes. Formation of coupled nanostructures is expected to result in enhanced local electric fields in the gap region, which consequently enhance the excitation efficiency of the fluorophores to lighten up the quenched fluorescence. The effects of different enhancing substrates and separation distances between fluorophore and the substrates were systematically investigated in the current study.

Scheme 1. Overall design of plasmon enhanced fluorescence by various coupled nanostructures.

Cy5 labelled ssDNA (oligo1 in Table 1) was chosen as the probe to study the enhancing capability of different metal NPs. 27 nm Au NSs (Figure 1A) were employed to quench the fluorescence of Cy5. Its absorption and emission spectra are shown in Figure S1A. Another ssDNA oligomer (target24) was employed as a linker to assemble oligo2 modified Au NSs and oligo3 modified enhancing substrates. 9 ACS Paragon Plus Environment


Oligo0 was utilized to control the amount of oligo1 on the surface of Au NSs. The SPR peak of the Au NSs became red-shifted by ~3 nm upon DNA modification (Figure S1B), indicating successful modification of metal NPs. After binding to Au NSs, fluorescence intensity of Cy5 was significantly quenched (Figure S1C), consistent with the previous report.29 6% of oligo1 labelled Au NSs (with fluorescence intensity quenched by 50-fold) were employed to study the effect of different substrates.

Figure 1. (A-C) TEM images and (D) normalized extinction spectra of Au NSs (27 nm, A), Au NRs (21×43 nm, B) and Au@Ag NSs (30 nm, C).

Fluorescence enhancement by different metal NPs Different metal NPs, Au NSs (~27 nm), Au NRs (21×43 nm) and Au@Ag NSs (~30 nm) have been utilized to turn on the quenched fluorescence of Cy5-Au NSs through Plasmon interactions. TEM images and extinction spectra of these three NPs are shown in Figure 1. Au NRs of 21×43 nm with SPR band maxima at 625 nm were selected because absorption/emission spectra of Cy 5 has good overlap with 10 ACS Paragon Plus Environment

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extinction spectra of coupled Au NRs and Au NSs, which is important for strong plasmonic interactions.30 Plasmon coupling interactions between noble metal NPs has been known to create hot spots with further enhanced local electric field, which is expected to cause significantly enhanced excitation efficiency and consequently enhanced fluorescence intensity. Successful coupling of Cy5-Au NSs with the enhancing substrates can be confirmed by monitoring their extinction spectra and TEM images of the assembled nanostructures (Figures S2 and S3). Using Au NRs as the illustration example, upon addition of DNA as the coupling agent to induce coupling interactions with Cy5-Au NSs, the SPR band at 500-650 nm steadily decreased, accompanied with increasing extinction at the longer wavelength region (700-1000nm) arising from the assembled nanostructures (Figure S2A). TEM images (Figures S2B and S3) indicate that assembled nanostructures are collective aggregates of multiple Au NRs and Au NSs lumped together, instead of simple 1:1 dimer-type assembly. These kinds of collective aggregates ensure strong plasmon coupling interactions between Cy5-Au NSs and the enhancing substrates. As shown in Figure 2, fluorescence intensity of Cy5-Au NSs was found to gradually increase upon coupling with Au NSs, Au NRs and Au@Ag NSs. The optimum fluorescence enhancement factors of 1.6, 1.8 and 7.9-fold were observed upon coupling with Au NSs, Au NRs and Au@Ag NSs, respectively. 30 nm Au@Ag NSs exhibited the highest fluorescence enhancement capability. The SPR signals of Au@Ag NPs primarily arise from the Ag component as Ag NPs have been known to act as more effective fluorescence enhancement substrates.31-32 11 ACS Paragon Plus Environment


Significantly enhanced fluorescence upon formation of nano-assembly arises from two factors. On the one hand, enhanced local electric field caused by coupling of metal NPs results in significantly enhanced excitation efficiency. On the other hand, formation of metal NPs aggregates results in redshifted extinction spectra. The redshifted extinction spectra of aggregated metal NPs have better spectral overlap with the emission spectrum of Cy5 molecules, which result in increased radiative decay rate of Cy5 and consequently improved quantum yield. In these aggregated metal nanostructures, both the excitation efficiencies and emission yield of Cy5 will be improved, which will result in a large enhancement factor in the overall fluorescence intensity.

Figure 2. Relative fluorescence intensity of Cy5-Au NSs upon coupling with different amount of Au NSs, Au NRs and Au@Ag NSs, respectively. The dash line labelled as 100% is fluorescence intensity of same amount of free Cy5.

Effects of dye distribution and size of the Au@Ag NSs However, the absolute fluorescence intensity is still far below that of the same amount of free Cy5 in solution (Figure 2). The unsatisfactory fluorescence enhancement can be ascribed to the drawback of the initial design (top panel in Scheme 2, a). In the 12 ACS Paragon Plus Environment

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initial design, Cy5 molecules are randomly distributed around Au NSs. Optimum enhancement effect generally requires the fluorophores to be located in the gap region of the coupled nanostructures. An improved design as shown in the bottom of Scheme 2 (b) is expected to give larger fluorescence enhancement. In this improved scheme, oligo5-labelled Cy5 was utilized to replace Cy5-oligo1 and oligo2 so as hybridization between oligomers to place Cy5 in-between Au NSs and Au@Ag NSs, where stronger local electric field amplification is expected to result in larger enhancement in the excitation efficiency and fluorescence intensity.33 Fluorescence of Cy5-oligo5 was quenched by 20-fold upon binding to the surface of Au NSs due to relative longer length of Cy5-oligo5 compared to Cy5-oligo1 that was used in the original design.20 Upon binding interactions with 30 nm Au@Ag NPs, fluorescence intensity of Cy5-Au NSs was enhanced by up to ~20-fold by adopting the new strategy (Scheme 2b) versus enhancement of 7.9-fold by using the old strategy (Scheme 2a).

Scheme 2. Improved scheme (from a to b) by placing Cy5 into the gap region of the coupled nanostructures.

Larger–sized metal NPs generally display stronger local electric field and potentially better fluorescence enhancement effect.34 When Au(17nm)@Ag(24nm) 13 ACS Paragon Plus Environment


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NSs (with an overall diameter of 65 nm, later we referred them as 65 nm Au@Ag NSs, see extinction spectrum and TEM images in Figure S4) were utilized as the enhancing substrate to couple with Cy5-Au NSs, fluorescence intensity of Cy5-Au NSs was enhanced by up to 100 fold, which is 5 times that of 30 nm Au@Ag NSs (Figure 3).

Figure 3. Relative fluorescence intensity enhancement of Cy5-AuNSs upon coupling interactions with Au@Ag NSs with diameters of 30 and 65 nm, and same amount of free oligo 5.

Effects of gap distance on fluorescence enhancement Metal enhanced fluorescence has been demonstrated to be strongly dependent on the separation distance between the chromophore and metal NPs.30-31,

35-38

Separation

distance dependent Plasmon induced fluorescence enhancement in the coupled nanostructures was investigated by controlling the gap distance between Au NSs and Au@Ag NSs using dsDNA of different lengths. Assuming a spacing of 0.34 nm between each base pair in dsDNA (Figure S5), three different separation distances (4.1, 8.2 and 12.2 nm) between Au NSs and Au@Ag NSs were obtained by using 12, 24 and 36 base pairs, respectively.39-40 Fluorescence intensities of Cy5-Au NSs were 14 ACS Paragon Plus Environment

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enhanced by up to 65, 80, and 48-fold upon coupling with 65 nm Au@Ag NSs for separation distance of 4.1, 8.2 and 12.2 nm, respectively (Figure 4). Optimum fluorescence enhancement of 80-fold was observed at an intermediate separation distance of 8.2 nm. The trend is consistent with the previous results that optimum enhancement generally occurred at the intermediate separation distance.31,

37

Less

fluorescence enhancement at longer separation distances can be ascribed to reduced electric field amplification,35,

41

while shorter separation distances will result in

fluorescence quenching effect due to energy transfer from the fluorophore to metal NPs.42 Plasmon coupling interaction in the coupled nanostructures was confirmed by the observation of redshifted and broadened extinction spectra upon formation of coupled nanostructures with different separation distances (Figure S6).

Figure 4. Separation distance dependent of fluorescence enhancement of Cy5-Au NSs upon coupling interactions with 65 nm Au@Ag NSs: 4.1, 8.2 and 12.2 nm.

Fluorescence lifetime measurements were performed to understand the enhancement mechanism of the coupled nanostructures. Fluorescence lifetimes of free Cy5-DNA, Cy5-Au NSs and Cy5 in coupled nanostructures with optimal fluorescence were measured by the TCSPC technique (Figure S7). The fluorescence lifetime of 15 ACS Paragon Plus Environment


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Cy5-DNA was 1.52 ns, which was shortened to 160 ps upon binding to the surface of Au NSs primarily due to increased nonradiative energy transfer process.28 However, fluorescence lifetimes of Cy5 in the coupled nanostructures with optimum fluorescence was further shortened and cannot be well resolved due to the limited temporal resolution of the TCSPC technique (~100 ps). The observation of significantly

increased

fluorescence

intensity

accompanied

with

shortened

fluorescence lifetime confirms that increased local electric field amplification in the coupled nanostructure, instead of releasing Cy5 from the surface of Au NS, is the major enhancement mechanism, which is consistent with the previous report.17

Applications in DNA Sequence Detection The above phenomenon of Plasmon enhanced fluorescence in the coupled nanostructures can be further utilized for potential applications in DNA sequence detection. As pre-quenched fluorescence offers reduced background, large Plasmon coupling induced fluorescence enhancement enables highly sensitive detection. The detection scheme is based on Scheme 2b. Upon addition of the target DNA, nanoassembly of oligo5-Au NSs and oligo3-Au@Ag NSs can be formed via DNA hybridization. As shown in Figure 5A, fluorescence intensity of the mixture steadily increased upon gradual addition of target DNA. Figure 5B shows fluorescence spectra at the low concentration range of target DNA. The change in fluorescence intensity can be clearly differentiated upon addition of target DNA with a concentration as low as 10 pM. Plot of fluorescence intensity versus concentration of 16 ACS Paragon Plus Environment

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target DNA shows that fluorescence intensity first increased rapidly upon addition of target DNA in the low concentration range, and then increase slowly before reaching a plateau at a concentration of 2.5 nM target DNA (Figure 5C). The limit of detection (LOD) was estimated to be 3.1 pM, which is lower than those of many other reported methods.43-44 The target DNA induced assembly process could also be monitored by the UV-vis spectra. Upon addition of target DNA, the original SPR peak at 450 nm gradually decreased while extinction in the longer wavelength region gradually increased (Figure S8). The extinction ratio at 660 and 450 nm (A660/A450) can also be employed to quantify the target DNA induced assembly (Figure S9). LOD based on the UV-vis method was estimated to be 60 pM, which is much higher than that of the fluorescence method (3.1 pM). The fluorescence method based on Plasmon coupling enhanced fluorescence gives much better sensitivity compared to the UV-Vis methods.

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Figure 5. (A, B) Fluorescence spectra of oliog5-Au NSs and oligo3-Au@Ag NSs in the presence of different concentrations of target DNA. (C) Plot of fluorescence enhancement factor versus concentration of target DNA. Inset is the low concentration range. (D) Fluorescence enhancement factors upon addition of 1 nM target DNA with different mismatched bases.

The selectivity of this method to detect perfectly matched DNA against mismatched ones has also been tested. As shown in Figure 5D, the perfectly matched DNA gave nearly 80-fold enhancement in fluorescence intensity, versus 8.1, 3.2 and 1.5-fold for one-, two-, and three-base mismatched ones, respectively. These results demonstrated that this method possesses high selectivity in differentiating perfectly matched DNA against mismatched ones. Single base mismatch can be easily distinguished even at the room temperature by using this Plasmon coupling enhanced fluorescence methods.

Conclusions Plasmon enhanced fluorescence in coupled nanostructures has been investigated by using a DNA based nano-assembly. The fluorescence of a model chromophore, Cy5, was pre-quenched by attaching to the surface of Au NSs through ssDNA. The quenched fluorescence of Cy5 was turned on by forming nano-assembly through DNA hybridization with different enhancing substrates: Au NSs, Au NRs and Au@Ag NSs. We have investigated the effects of several factors, including composition, shape and size of metal NPs, the distribution of the dye molecules, as well as the gap distance between coupled NPs. Among three different metal NPs, Au@Ag NSs were 18 ACS Paragon Plus Environment

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found to display largest fluorescence enhancement effects. Larger sized Au@Ag NPs were found to display larger fluorescence enhancement effects compared to smaller ones. When 65 nm Au@Ag NPs were utilized as the enhancing substrate, the optimum enhancement effects were observed at an intermediate separation distance of 8.2 nm between Au NSs and Au@Ag NSs. The optimum fluorescence enhancement was up to 100-fold compared to quenched Cy5-Au NSs, 5-fold compared to unquenched free Cy5 molecules. This Plasmon coupling enhanced fluorescence phenomenon has been further explored as a simple turn-on platform for highly sensitive and selective detection of DNA sequence. The LOD of this method for DNA sequence detection was estimated to be 3.1 pM, much better than the UV-Vis methods under the similar experimental conditions. The exceptional selectivity of this method allow detection of single-base mismatch at the room temperature. This Plasmon coupling enhanced fluorescence method could be further utilized to develop various platforms for highly sensitive chemical and biological sensing and imaging applications.

Supporting Information Absorption and fluorescence spectra of Cy5; TEM images and UV-Vis extinction spectrum of Au@Ag NSs (65 nm); fluorescence spectra of 6% oligo1 labeled 27 nm Au NSs before and after treatment with DTT; extinction spectra and TEM images of Au NRs upon addition of Cy5-Au NSs; extinction spectrum and TEM image of 65 nm Au@Ag NSs; scheme of coupled nanostructures of different separation distances; extinction spectra of coupled AuNS-Au@AgNS nanostructures with different separate distances; fluorescence lifetime measurement results; plot of extinction ratio at 660 19 ACS Paragon Plus Environment


and 450 nm of coupled nanostructures versus the concentration of target DNA. This material is available free of charge via the Internet at http://pubs.acs.org.

Corresponding Authors *E-mail: [email protected]. Fax: +65 6779-1691. Tel: +65 6516-7880 (Q.-H. Xu). The authors declare no competing financial interest.

Acknowledgement We thank the financial support from the Ministry of Education, Singapore (Grant number R-143-000-607-112), and the National Research Foundation, Prime Minister’s Office, Singapore under its Competitive Research Program (CRP Award No. NRF-CRP10-2012-04).

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TOC


Plasmon Enhanced Fluorescence in Coupled Nanostructures and

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