Approach for Plasmonic Based DNA Sensing: Amplification of the

Article pubs.acs.org/ac

Approach for Plasmonic Based DNA Sensing: Amplification of the Wavelength Shift and Simultaneous Detection of the Plasmon Modes of Gold Nanostructures Jolanda Spadavecchia,*,† Alexandre Barras,‡ Joel Lyskawa,§ Patrice Woisel,§ William Laure,§ Claire-Marie Pradier,† Rabah Boukherroub,† and Sabine Szunerits*,† †

Laboratoire de Réactivité de Surfaces, UMR CNRS 7197, Université Pierre & Marie Curie − Paris VI, Site d’Ivry − Le Raphaël, 94200 Ivry-sur-Seine, France ‡ Institut de Recherche Interdisciplinaire (IRI, USR 3078 CNRS), Université Lille 1, Parc de la Haute Borne, 50 Avenue de Halley, BP 70478, 59658 Villeneuve d’Ascq, France § Université Lille 1, Unité des Matériaux Et Transformations (UMET, UMR 8207 CNRS) Ingénierie des Systèmes polymères (ISP) Team, F-59655 Villeneuve d’Ascq Cedex, France ABSTRACT: In this article, the detection of DNA hybridization taking advantage of the plasmonic properties of gold nanostructures is described. The approach is based on the amplification of the wavelength shift of a multilayered localized surface plasmon resonance (LSPR) sensor interface upon hybridization with gold nanorods and nanostarslabeled DNA. The amplification results in a significant decrease of the limit of detection from ≈40 nM as observed for unlabeled DNA to 0.2 nM for labeled DNA molecules. Furthermore, the plasmonic band, characteristic of the labeled DNA, is different from that of the LSPR interface. Indeed, next to the plasmon band at around 550 nm, being in resonance with the plasmon band of the LSPR interface, additional plasmonic peaks at 439 nm for gold nanostar-labeled DNA and 797 nm for gold nanorod-labeled DNA are observed, which were used as plasmonic signatures for successful hybridization.

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These interfaces were obtained by thermal evaporation of thin (2−4 nm) gold or silver films on glass and postannealing at elevated temperature. The thermal treatment results in the formation of individual spheroidal to ellipsoidal shaped particles. The particles were postcoated with nanometer thick dielectric films to overcome the poor adhesion of the metal island films to the substrate, yielding stable LSPR interfaces. In an attempt to enhance the sensitivity of multilayered LSPR interfaces for the detection of hybridization events, a highly sensitive detection of DNA hybridization was achieved using metal nanostructures enhanced fluorescence.6 Fluorescently labeled DNA oligomers were covalently immobilized on a thin amorphous silicon−carbon layer capping the gold metal nanostructures. Through optimization of the coating thickness and by working close to resonance condition for plasmon and fluorophore excitation, the hybridization of very dilute (5 fM) oligomers was easily attained. In this article, we describe a different approach for an easy and sensitive detection of DNA hybridization. It is based on the plasmonic coupling between gold nanostructures on the LSPR

NA hybridization assays are widely employed in molecular biology and for forensic tests.1 Traditionally, DNA assays are based on fluorescent read out by labeling the target DNA with a fluorescent molecule.2 Biosensors taking advantage of the plasmonic properties of metal films and nanostructures have emerged as alternative methods for detecting DNA hybridization with lower cost and high sensitivity.3−7 Localized surface plasmon resonance (LSPR) sensors, employing noble metal nanostructures, have lately attracted considerable attention as a new class of plasmonic nanosensors.8,9 The exceptional optical properties of the metallic nanostructures result from the participation of the particles’ free electrons in the collective oscillation of electrons with a plasmon resonance frequency occurring in the visible range. The position of the nanoparticles extinction maximum (λmax) is highly dependent on the local refractive index of the nanoparticles, and biomolecular binding/unbinding events can be followed in real-time by monitoring the temporal variation in the LSPR signal. However, sensitivity loss can arise when LSPR sensors are functionalized with long surface ligands,10 as is the case for the linking of oligonucleotides to LSPR interfaces. In recent reports, we have shown that detection limits in the nanomolar range (2−60 nM) can be achieved for DNA hybridization using multilayered LSPR interfaces.5−7,11 © 2013 American Chemical Society

Received: December 14, 2012 Accepted: February 15, 2013 Published: February 17, 2013 3288

dx.doi.org/10.1021/ac3036316 | Anal. Chem. 2013, 85, 3288−3296

Analytical Chemistry

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The hybridization buffer was a solution of NaCl (0.5 M), phosphate buffer solution (0.01 M), and ethylenediaminetetraacetic acid (0.01 M, pH 5.5). Synthesis of Maleimide-Terminated Dopamine. Maleimide-terminated dopamine was used to link oligonucleotide probes to the SiO2 coated nanostructured surfaces. Maleimideterminated dopamine was synthesized according to ref 16. Dopamine hydrochloride (1.92 g, 0.010 mol) and 1.28 g of triethylamine (0.013 mol) dissolved in anhydrous methanol (10 mL) were added dropwise to 6-maleimidohexanoic acid Nhydroxysuccinimide ester (2.6 g, 0.008 mol) in anhydrous CH2Cl2 (100 mL). The reaction was stirred vigorously for 48 h under nitrogen. The solvents were evaporated under reduced pressure, and the residue was dissolved in dichloromethane (100 mL). The organic phase was washed three times with HCl (0.5 M, 80 mL) and dried over MgSO4. After filtration, the solvent was evaporated and the crude product was purified by column chromatography using CH2Cl2/MeOH (10:1) as eluent. The product was obtained as a yellow solid in 30% yield. Formation of Gold Nanoislands on Glass (Au NIs/ Glass). Glass slides (76 × 26 × 1 mm3) were first cleaned in isopropanol and acetone in an ultrasound bath at room temperature, rinsed copiously with Milli-Q water, and dried under a stream of nitrogen. The clean substrates were then transferred into an evaporation chamber. Gold island deposition was carried out by thermal evaporation of 4 nm thick gold films using MEB 550 S (Plassys, France). Postdeposition annealing of the Au-covered slides was carried out at 500 °C for 1 min under nitrogen atmosphere using a rapid thermal annealer (Jipelec Jet First 100) (short hot thermal annealing). The reproducibility of the Au evaporation was evaluated by measuring the LSPR signals of a batch of 8 samples. The standard deviation in the wavelength (λmax) and maximum absorption (Imax) is typically 2 nm and 0.02 abs units, respectively. Deposition of SiOx Overlayers. SiOx overlayers were deposited on glass coated with Au nanoislands by plasmaenhanced chemical vapor deposition (PECVD) in a Plasmalab 800Plus (Oxford Instruments, UK) at a pressure of 0.005 Torr for 1 h. The growth conditions used were as follows: substrate temperature, 300 °C; gas mixture, SiH4 (3% in N2) and N2O (the gas flow was 260 and 700 sccm for SiH4 and N2O, respectively); total pressure in the reactor, 1 Torr; power, 10 W at 13.56 MHz. Under these experimental conditions, the deposition rate was 414 Ǻ min−1 and the silica films display a refractive index of 1.48. A 7 nm thick film of SiOx was deposited on the glass/Au nanoislands interfaces. Oligonucleotide Linking to Glass/Gold Nanoislands/ Silicon Dioxide (Glass/Au NIs/SiOx) Interface. Before reaction, the glass/Au NIs/SiOx interfaces were first cleaned by UV/ozone to remove any organic contaminants on the surface and to generate surface hydroxyl groups. The linking of maleimide-terminated dopamine on the SiOx overlayer was performed by immersing the interface into dopamine solution (10 mM in acetonitrile) and sonicating for 8 h at room temperature. The resulting surface was washed copiously with acetonitrile and methanol and dried under nitrogen stream. The following protocol was used to link DNA molecules (probe 1) on the maleimide-terminated surface. Twenty μL of DNA probe 1 (100 nM in PBS buffer (0.1 M, pH 7.4)) was added onto a maleimide-terminated interface. A coverslip was placed on top of the drop and allowed to incubate for 3 h at 4

interface and gold nanorods (Au NRs) or gold nanostars (Au NSs) modified DNA molecules in solution. The plasmonically active nanotructured labels are used in two different ways. On the one hand, the plasmon band of the nanostructures is close to that of the LSPR interface, resulting in resonant coupling which enhances the resonance wavelength shift upon hybridization. On the other hand, the nanostructures in solution show additional plasmonic bands well separated from that of the resonance maximum of LSPR. Following the appearance of this additional plasmon band upon hybridization, the detection of a successful hybridization event is allowed in an easy and accurate manner. The use of plasmonically active nanostructures as labels modified with antibodies has indeed been recently shown to improve the sensitivity of plasmon based bioassays, where a biotin−antibiotin binding event was used as a model.12 A similar enhancement was demonstrated earlier using gold nanoparticles labeled biotin molecules on a streptavidinfunctionalized LSPR interface.13 More recently, some of us demonstrated that the formation of well dispersed colloidal solutions of gold nanorods after bioconjugation to antibodies is benefical for the enhancement of the sensitivity of a Fouriertransform surface plasmon resonance (FT-SPR) biosensor.14 The use of gold nanorods rather than nanospheres has in this context proven to be highly attractive, as the plasmon wavelength can be tuned over a great wavelength range by varying the size and aspect ratio.14,15 This article widens the concept of enhancing the sensitivity of plasmonic sensors with nanolabels in solution to DNA hybridization assays. More importantly, the interest of a careful design of the nanostructures used for signal amplification is demonstrated. In the present work, gold nanorods and gold nanostars have been synthesized and used to label DNA probes. Hybridization was assayed on multilayered LSPR surfaces, making clear a remarkable signal enhancement as well as new absorption bands when targets were labeled with these original gold nanostructures.

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EXPERIMENTAL SECTION Materials. All chemicals were reagent grade or higher and were used as received unless otherwise specified. Sodium hydroxide (NaOH), sodium dodecyl sulfate (SDS), salmon sperm DNA, formamide, phosphate buffer saline (PBS, 0.1 M, pH 7.4), acetone, acetonitrile (CH3CN), methanol (CH3OH), cetyltrimethyl ammonium bromide (CTAB), tetrachloroauric acid (HAuCl4), silver nitrate (AgNO3), sodium borohydride (NaBH4), ascorbic acid, Protoporphyrin IX (95%), and ethanol (C2H5OH) were purchased from Sigma Aldrich. Saline sodium citrate buffer (SSC) was obtained from Fluka. The 40mer oligonucleotides were purchased from Eurogentec and have the following sequences. DNA on LSPR interface (probe 1): 5′-SH-TTT-TTTTTT-TTT-TTT-TAT-TCT-TCT-GGA-CTA-TCG-ATCGCT-T-3′ Complementary DNA on gold nanoparticles (probe 2): 5′SH-TTT-TTT-TTT-TTT-TTT-AAG-CGA-TCG-ATA-GTCCAC-AAG-AAT-A-3′ Noncomplementary DNA on gold nanoparticles (3-base mismatch, probe 3): 5′-SH-TTT-TTT-TTT-TTT-TTT-TATTCT-TCT-GGA-ATA-ACG-ATC-ACT-T Probe in solution (probe 4): AAG-CGA-TCG-ATA-GTCCAG-AAG-AAT-A Stock solutions of 1 μM were prepared in PBS buffer (100 mM, pH 7.4) containing 0.01% sodium dodecyl sulfate (SDS). 3289



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°C. The DNA-immobilized interface was washed three times in water and then immersed in water for subsequent hybridization. Synthesis of Gold Nanostars and Gold Nanorods. Gold nanorods were made and purified following the well established seed-mediated procedure described in previous papers.17 Gold seeds of around 3−4 nm, protected by CTAB, were prepared by reduction of the required amounts of Au3+ ions by ice− cooled NaBH4 (0.01 M; 0.6 mL) in the presence of CTAB (0.20 M; 5 mL). After 4 h, a calculated amount of seed solution was added to a growth solution containing CTAB (5 mL; 0.20 M), HAuCl4 (5 mL; 1 x 10−3 M), AgNO3 (0.25 mL; 4 × 10−3 M), and ascorbic acid (70 μL; 8 × 10−3 M). The as-prepared nanorod solution was centrifugated at 11.000 rpm for 26 min for three times, and then, the supernatant was discarded and the residue was redispersed in an equivalent amount of buffer solution (PBS pH: 7). This was repeated twice principally to remove excess of CTAB. Stock solutions were stored at 27−29 °C and characterized using UV−vis spectroscopy and transmission electron microscopy (TEM). Gold nanostars were obtained when Protoporphyrin IX was added both in the seed and in the growth solutions, following the seed-mediated procedure. Briefly, gold seeds were prepared by adding a given quantity of Protoporphyrin IX into the reaction mixture. A volume of 5 mL of 0.20 M CTAB was added to 5 mL of 6.29 × 10−4 M of Protoporphyrin IX solution prepared by adding 17.7 mg of Protoporphyrin IX in EtOH/ H2O (3:2) solution at 27 °C under stirring conditions; this Protoporphyrin IX concentration was chosen because it led to the best resolved UV−visible spectrum of the Protoporphyrin IX solution. Five mL of an aqueous solution containing 2.5 × 10−4 M HAuCl4 and 0.6 mL of ice−cooled NaBH4 (0.01 M) were then added. After 4 h, a calculated amount of seed solution (12 μL) was added to a growth solution containing CTAB (5 mL; 0.20 M), Protoporphyrin IX (5 mL; 29 × 10−4 M), HAuCl4 (5 mL; 1 x10−3 M), AgNO3 (0.25 mL; 4 × 10−3 M), and ascorbic acid (70 μL; 8 × 10−3 M). The gold nanostar solution was centrifugated at 11.000 rpm for 26 min for three times, and then, the supernatant was discarded and the residue was redispersed in an equivalent amount of buffer solution (PBS pH: 7). This was repeated twice principally to remove excess of CTAB. Stock solutions were stored at 27−29 °C and characterized using UV−vis spectroscopy and transmission electron microscopy (TEM). Bioconjugation of DNA to Nanorods and Nanostars. Gold nanorods (Au NRs) and gold nanostars (Au NSs) were chemically modified with a 5′-thiol capped 40 base oligonucleotides (probe 2 or 3) according to the procedure described by Mirkin et al.18 All oligonucleotides used in this study were synthesized on the basis of a previously characterized plant gene. In particular, our study is based on the A. carbonarium DNA sequences involved in the biosynthesis of Tricohothecenes mycotoxins. To 200 μL of Au NRs or Au NSs solution (20 nM in 0.1 M PBS) was added 25 μL of 100 nM HS-DNA (probe 2 or 3) solution. After standing for 16 h, the solution was mixed with 0.25 mL of 10% NaCl. Next, the gold nanostructures/HS-DNA was centrifuged twice at 6000g for 20 s to remove HS-DNA, and pellets were redispersed in PBS buffer (1 M NaCl, 100 mM phosphate buffer, pH 7). The resultant colloidal solution was sonicated for 5 min and then stirred for 1 h at room temperature. DNA Hybridization. For end-point measurement, the surface was exposed to the complementary targets (labeled and nonlabeled) at 42 °C during 4 h in a hybridization

chamber. The hybridization solutions were made of 2× SSC, 0.1% SDS, 0.1% salmon sperm DNA, 35% formamide, and the target oligonucleotides at a concentration chosen in the range of 20 pM to 100 nM. After hybridization, the sample was submitted to three successive wash steps of 2 min each in the following solutions: 2× SSC, 0.1% SDS, then 1× SSC, 0.1% SDS, and finally 0.1× SSC, all at pH 7. The sample was finally dried under a stream of argon before LSPR measurement. The denaturation of hybridized DNA was performed using NaOH (0.1 M) during 2 min followed with rinsing with deionized water. Instrumentation. UV/Vis Measurements. Absorption spectra were recorded using a Perkin-Elmer Lambda UV/vis 950 spectrophotometer in plastic cuvettes with an optical path of 10 mm. The wavelength range was 400−700 nm or 400− 1100 nm. Scanning Electron Microscopy (SEM). SEM images were obtained using an electron microscope ULTRA 55 (Zeiss, France) equipped with a thermal field emission emitter and three different detectors (EsB detector with filter grid, high efficiency In-lens SE detector, and Everhart-Thornley Secondary Electron detector). Transmission Electron Microscopy (TEM). Transmission electron microscopy measurements were performed with a JEOL JEM 1011 microscope operating at an accelerating voltage of 100 kV. The TEM images were taken after separating the surfactant from the metal particles by centrifugation. Typically, 1 mL of the sample was centrifuged for 21 min at a speed of 11.000 rpm. The upper part of the colorlesss solution was removed, and the solution fraction was redispersed in 1 mL of buffer solution (PBS, pH 7). Two μL of this redispersed particle suspension was placed on a carbon-coated copper grid and dried at room temperature. X-ray Photoelectron Spectroscopy. X-ray photoelectron spectroscopy (XPS) measurements were performed with an ESCALAB 220 XL spectrometer from Vacuum Generators featuring a monochromatic Al Kα X-ray source (1486.6 eV) and a spherical energy analyzer operated in the CAE (constant analyzer energy) mode (CAE = 100 eV for survey spectra and CAE = 40 eV for high-resolution spectra), using the electromagnetic lens mode. The detection angle of the photoelectrons is 30°, as referenced to the sample surface. The XPS spectra were corrected according to the binding energies of Au 4f7/2, equal to 80.0 eV. Contact Angle Measurements. Water contact angles were measured using deionized water. We used a remote-computer controlled goniometer system (DIGIDROP by GBX, France) for measuring the contact angles. The accuracy is ±2°. All measurements were made in ambient atmosphere at room temperature.

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RESULTS AND DISCUSSION Formation of Gold Nanostars and Gold Nanorods Modified with Thiolated-DNA. Gold nanorods (Au NRs) were fabricated by a seed-mediated procedure, where gold seeds of 3−4 nm in diameter protected by cetyltrimethyl ammonium bromide (CTAB) where first prepared by reduction of tetrachloroauric acid (HAuCl4). Growth of the seeds was achieved by adding silver nitrate and ascorbic acid to the growth solution. The synthesis of gold nanostars (Au NSs) was achieved by mixing porphyrin molecules (Protoporphyrin IX) with CTAB as surfactants and/or shape-modulating agents.19 In addition, a large quantity of Protoporphyrin IX 3290



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Figure 1. TEM images of gold nanorods (Au NRs) (A) and gold nanostars (Au NSs) (B). LSPR spectra of Au NRs (C) and Au NSs (D) in water before (black) and after (blue) conjugation with thiolated DNA (probe 2).

branches with small overall size variation shift the plamon bands to the red.26 The Au NSs formed exhibit in our case extinction bands at 402 nm, due to interband transaction,27−29 and at 587 nm (Figure 1D), due to the rather short branches. The colloidal gold solutions were incubated with thiolatedDNA (probe 2, 3) for 16 h to allow conjugation to occur. To verify that the DNA has been successfully attached to the gold nanostructures, UV/vis measurements were performed after the modification step (Figure 1C,D). In the case of Au NRs, the extinction bands at λmax,1 = 548 nm and λmax,2 = 706 nm shifted to 570 nm (Δλmax = 22 nm) and 744 nm (Δλmax = 38 nm), respectively. The larger shift of the second extinction band is believed to be correlated with higher refractive index sensitivity of this plasmon band.9 In general, red-shifted plasmon bands have longer electromagnetic field decay lengths and therefore higher sensitivies.30 In the case of Au NSs, the plasmon band at 402 nm shifts to 427 nm (Δλmax = 25 nm) but looses strongly in intensity, while the broad band with two maxima at 548 and 587 nm shifts to a single band at 608 nm. Modification of LSPR Interface with Thiolated-DNA. To demonstrate the ability of DNA modified gold nanostructures to bind specifically to complementary oligonucleotides and enhance the LSPR signal, an LSPR interface consisting of gold nanoislands (Au NIs), deposited on glass slide, coated with a thin silica film was prepared. The fabrication process comprises the following steps: (i) thermal evaporation of 4 nm

in the seed and growth solutions produces star-shaped particles of about 50 nm in the longest direction. As previously shown by some of us, the presence of Protoporphyrin IX in the growth solution most likely improves the reduction process and promotes the development of side branches.19 This approach differs to other reported ones, where CTAB was partially replaced with CTAC (cetyltrimethylammonium chloride) by adjusting the NaBr concentration in the growth solution.20−22 Figure 1A shows a TEM image of the Au NRs after deposition on a microscope grid. The Au NRs are well dispersed in size and shape with an average length of 33 ± 1 nm and width of 13 ± 1.8 nm, estimated from ≈350 rods on a given TEM image. Figure 1B is a representative TEM image of gold nanoparticles prepared by addition of Protoporphyrin IX both in the seed and in the growth solutions; one observes a change of shape, from rods to stars of about 50 nm. The extinction spectrum recorded on a colloidal solution of nanorods is presented in Figure 1C and shows two extinction bands at λmax,1 = 548 nm and λmax,2 = 706 nm. The resonance band at 706 nm corresponds to the longitudinal plasmon oscillations, while the band at 548 nm is due to the transverse plasmon oscillation band, confirming the presence of elongated Au NRs. The optical properties of Au NSs are known to be highly anisotropic and to strongly depend on the size of the protruding multiple sharp branches that act as hot zones to greatly enhance the local electromagnetic field.23−25 Longer, sharper, and more 3291



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gold followed by hot annealing at 500 °C under argon atmosphere for 1 min,9,11 (ii) coating with SiOx thin film. The morphology of the resulting nanoislands has been investigated previously by us. Scanning electron microscopy (SEM) shows nanoislands with an average diameter of 25 ± 5 nm, a height of 14 ± 3 nm, and an average particle−particle distance of 45 ± 10 nm (Figure 2A).31,32 The thermal treatment results in the

There are also some reports on its use for the functionalization of nonmetal oxides such as SiO2.39,40 The attachment chemistry based on a maleimide-modified dopamine onto the LSPR interface was thus developed. Such dopamine derivatives prevent self-oxidation and polymerization, due to the absence of free amine groups, and mostly form monolayers on the substrate. A detailed characterization of the surface composition after modification of the LSPR interface with maleimide-terminated dopamine was performed by X-ray photoelectron spectroscopy (XPS). The XPS survey spectrum of the modified interface shows peaks due to Au4f (82 eV), Si2p (101 eV), Si2s (153 eV), C1s (285 eV), Au4d (334 and 353 eV), N1s (400 eV), and O1s (530 eV) in accordance with the chemical composition of the surface. The ratio of C/N is found to be experimentally 8.6, somewhat larger than the theoretical value of 6. The larger amount of carbon is linked to some surface contamination. A good estimation of the success of the surface reaction can be seen in the relatively broad (>1.5 eV) N1s band at 400.1 eV due to the presence of OC−N− and NH−CO functional groups (Figure 3B). Immobilization of thiolated-DNA (probe 1) to the LSPR interface was achieved through reaction of the maleimide function with the thiol groups borne by the DNA molecules (Figure 3A). The reaction between thiols and maleimides is commonly applied in the field of bioconjugation and thus maintains the acceptability of the process of biological applications.41−43 In this reaction, the thiol is added across the double bond of the maleimide to yield a thioether.42 The covalent linking of the thiolated DNA stand is unambiguously evidenced by the presence of the S2p band in the XPS survey spectrum (Figure 3C). The high resolution XPS of the S2p band shows contributions at 164.2 eV (S2p3/2) and 165.4 eV (S2p1/2), characteristic of C−S bonds and small peaks at 167.1 (S2p3/2) and 168.2 eV (S2p1/2) from some oxidized sulfur. The extinction spectrum of the DNA-modified LSPR sensor was recorded prior to the incubation with complementary DNA (Figure 2B) and showed a band at 579 nm, 15 nm red-shifted when compared to unmodified glass/Au NIs/SiOx. The plasmonic shift is lower than in the case of gold nanostars and nanrods before and after DNA conjugation, where a red shift of ≈20 nm is observed (Figure 1C,D). The difference might be correlated to the lower refractive index sensitivity of the plasmonic interface when compared to the nanostructures in solution or due to different orientations of the DNA strands on the interface. Hybridization Experiments. To demonstrate the ability of the DNA molecules modified with gold nanostructures to bind specifically to an LSPR interface modified with its complementary DNA strand and to enhance the hybridization signal, the LSPR interface was incubated for 4 h at 37 °C with unlabeled complementary DNA (probe 4) and complementary DNA labeled with Au nanostructures (probe 3) (Figure 4A). Following the hybridization, the extinction spectrum of the LSPR sensor was again recorded. For a 100 nM unlabeled complementary DNA, a shift of λmax of 6 nm to the red was recorded (Figure 4B). To see how this value compares to the case of nanorods (Figures 4C) or nanostars-labeled DNA (Figures 4D), the same experiment was performed replacing the unlabeled DNA with Au nanostructure-labeled DNA (100 nM). In the case of Au NRs labeled DNA, hybridization results in a shift of the plasmon band by 43 nm with the appearance of an additional band at 797 nm, characteristic of the nanorod label. Comparable results are observed with Au NSs labeled

Figure 2. (A) SEM image of glass/Au NIs/SiOx interface obtained through thermal deposition of 4 nm thick gold film on glass followed by postannealing at 500 °C for 1 min under argon. (B) UV/vis transmission spectra of glass/Au NIs (black), glass/Au NIs/SiOx (blue), and glass/Au NIs/SiOx (green) modified with single strand DNA molecules, probe 1.

coalescence of aggregated gold nanoislands on the glass interface and sharper LSPR signal with λmax = 552 nm (Figure 2B). To stabilize the Au NIs and to allow the covalent linking of oligonucleotide units onto the LSPR interface, a 7 nm thick silica film was further deposited onto the Au NIS using chemical vapor decomposition of a gas mixture of SiH4 and N2O in a plasma reactor at 300 °C.33 The resulting multilayered LSPR surface showed a refractive index sensitivity of S = 70 nm/RIU (refractive index units), slightly lower than the uncoated one with a S = 96 nm/RIU.31 Figure 2B displays the UV transmission spectra of the glass/Au NIs/SiOx interface. As expected, the LSPR spectrum shows a red-shift in the presence of 7 nm SiOx to λmax = 564 nm.11,33 This interface was, in the following, modified with DNA (probe 1) molecules. The surface modification route is outlined in Figure 3A. Indeed, depending on the substrate to be modified, different surface chemistries have been established. In the case of silicon dioxide, alkylsilanes are most widely employed.34,35 While silane molecules have good physical stability, they have the disadvantage of being sensitive to moisture. Furthermore, the formation of monolayers is rather difficult for short chain silanes such as 3-aminopropyltriethoxysilane. Recently, dopamine and its derivatives have been exploited as a novel anchoring material on metal, metal oxide, and diamond interfaces.36−38 3292



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Figure 3. (A) Schematic illustration of the immobilization of 20mer ODN on glass/Au NIs/SiOx interfaces. 1: linking of maleimide−dopamine; 2: reaction of maleimide function with thiolated DNA molecules (probe 1). (B) N1s high resolution XPS spectrum of glass/Au NIs/SiOx interface functionalized with maleimide−dopamine. (C) high resolution XPS spectrum of S2p of glass/Au NIs/SiOx modified with thiolated DNA (probe 1).

easy signature of the success of the hybridization event. The change of the absorption intensity at 797 nm (Au NRs labeled DNA) and 429 nm (Au NSs labeled DNA) upon DNA hybridization with varying concentrations is displayed in Figure 5B and indicates a detection limit of 400 pM for Au NRs and 20 nM for Au NSs. The lower detection limit of the band at 797 nm might be related to the higher sensitivity to the refractive index change of this plasmon band.

DNA. As seen in Figure 4D, hybridization resulted in a shift of the plasmon band of the interface by 41 nm. Moreover, the presence of a second band at 429 nm, due to the plasmon band of the nanostars becomes obvious. A nonspecific binding experiment, in which 100 nM Au NRs and Au NSs labeled with a DNA sequence with three mismatches (probe 3) was exposed to the LSPR interface, showed a minimal change in λmax (3−7 nm) with no clear appearance of the band of the gold nanolabels at 429 and 797 nm, respectively. To determine the dynamic range of this signal amplification, the LSPR shift upon DNA hybridization for several concentrations of DNA and Au nanostructures labeled complementary DNA was measured (Figure 5A). With a minimum detectable Δλmax of 0.35 nm, the limit of detection was ≈200 pM for DNA labeled with Au NRs or NSs, while it was ≈40 nM for unlabeled DNA. The decrease of the detection limit is believed to be primarily related to the plasmon coupling effects. However, it might be also strongly influenced by the multivalency effects as each DNA binding event between the labeled DNA and the LSPR interface is most likely mediated by several DNA stands on the particle. Further studies need to be undertaken to ascertain this effect. However, in addition to measuring the change in the position of λmax of the LSPR interface upon DNA binding events, the appearance of the additional plasmonic band due to the presence of the nanostructures labels can also be used as an

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CONCLUSION We demonstrated that the detection limit of LSPR based DNA sensors can be improved in two different ways. The use of DNA labeled with Au NRs and Au NSs allows one to increase drastically the observed response from LSPR sensors. This increase shifts the limit of detection to 200 pM, a large improvement compared to unlabeled DNA with a LOD of 40 nM. The observed enhancement in the LSPR shift is produced by plasmonic coupling between the Au nanostructures labels and the Au islands on the LSPR interface. The shape of the nanostructures seems not to influence strongly the degree of coupling as seen in a similar shift of the LSPR sensors wavelength upon DNA hybridization. However, the structure of the labels becomes of interest if its plasmonic characteristics are used for the detection of a successful hybridization event. Indeed, the appearance of additional plasmonic peaks at 439 nm for Au NSs and 797 nm for Au NRs labeled DNA can serve 3293



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Figure 4. (A) Schematic illustration of the hybridization process of the DNA-functionalized LSPR chip with unlabeled complementary DNA and gold-nanostructures labeled DNA molecules. (B) LSPR spectra before (black) and after (blue) incubation with 100 nM unlabeled complementary DNA (probe 4). (C) LSPR spectra before (black) and after (green) incubation with complementary DNA labeled with Au NRs (probe 2). (D) LSPR spectra before (black) and after (red) incubation with complementary DNA labeled with Au NSs (probe 2). (E) Nonspecific binding assay to test nonspecific interaction of the LSPR chip functionalized with DNA molecules (probe 1) with Au NSs only (blue), Au NRs (green), and noncomplementary DNA molecules (red).

to sense the DNA hybridization event. Detection of the appearance of the plasmon band at 797 nm upon DNA

hybridization resulted in LOD of 400 pM, comparable with the signal enhanced mode. It is thus considered as a new and 3294



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Figure 5. Quantitative LSPR response curves for different concentrations of unlabeled complementary DNA (blue), Au NRs labeled complementary DNA (red) and Au NSs labeled complementary DNA (green). (A) Shift of λmax of LSRP interface upon hybridization events. (B) Appearance of the bands at 429 nm (Au NSs) and 797 nm (Au NRs). (13) Haes, A. J.; Van Duyne, R. P. J. Am. Chem. Soc. 2002, 124, 10596. (14) Spadavecchia, J.; Casal, S.; Boujday, S.; Pradier, C.-M. Colloids Surf., B: Biointerfaces 2012, 100, 1. (15) Burda, C.; Chen, X. B.; Narayanan, R.; El-Sayed, M. A. Chem. Rev. 2005, 105, 1025. (16) Mazur, M.; Barras, A.; Kuncser, V.; Galatanu, A.; Zaitzev, V.; Woisel, P.; Lyskawa, J.; Laure, W.; Siriwardena, A.; Boukherroub, R.; Szunerits, S. Nanoscale 2013, DOI: 10.1039/C3NR33506B. (17) Jana, N. R.; Gaearheart, L.; Murohy, C. J. Adv. Mater. 2001, 13, 1389. (18) Mirkin, C. A.; Letsinger, L. R.; Mucic, C. R.; Storhof, J. J. Nature 1996, 382, 607. (19) Spadavecchia, J.; Casale, S.; Landoulsi, J.; Pradier, C.-M. Nanotechnology, 2013, submitted ref: NANO/464583/PAP/303527. (20) Fan, F.-R.; Liu, D.-Y.; Wu, Y.-F.; Duan, S.; Xie, Z.-X.; Jiang, Z.Y.; Tian, Z.-Q. J. Am. Chem. Soc. 2008, 130, 6949−6951. (21) Pande, S.; Ghosh, S. K.; Praharaj, S.; Panigrahi, S.; Basu, S.; Jana, S.; Pal, A.; Tsukuda, T.; Pal, T. J. Phys. Chem C 2007, 111, 10806. (22) Wu, H.-L.; Chen, C.-H.; Huang, M. H. Chem. Mater. 2008, 1, 110. (23) Hao, F.; Nethel, C. L.; Hafner, J. H.; Nordlander, P. Nano Lett. 2007, 7, 729. (24) Nehl, C. L.; Liao, H. W.; Hafner, J. H. Nano Lett. 2006, 6, 683. (25) Ahmed, W.; Kooij, E. S.; van Silfhout, A.; Poelsema, B. Nanotechnology 2010, 21, 125605. (26) Yuan, H.; G., K. C.; Hwang, H.; Wilson, C. M.; Grant, G. A.; Vo-Dinh, T. Nanotechnology 2012, 23, 075102. (27) Zach, M.; Kasemo, B.; Langhammer, C. ACS Nano 2011, 5, 2535. (28) Dulkeith, E.; Niedereichholz, T.; Klar, T. A.; Feldmann, J.; von Plessen, G.; Gittins, D. I.; Mayya, K. S.; Caruso, F. Phys. Rev. B 2004, 70, 205424. (29) Beversluis, M. R.; Bouhelier, A.; Novotny, L. Phys. Rev. B 2003, 68, 115433. (30) Anker, J. N.; Paige Hall, W.; Lyandres, O.; Shah, N. C.; Zhao, J.; Van Duyne, R. P. Nat. Mater. 2008, 7, 442. (31) Galopin, E.; Niedziółka-Jönsson, J.; Akjouj, A.; Pennec, Y.; Djafari-Rouhani, B.; Noual, A.; Boukherroub, R.; Szunerits, S. J. Phys. Chem. C 2010, 114, 11769. (32) Szunerits, S.; Boukherroub, R. Global J. Phys. Chem. 2010, 1, 20. (33) Szunerits, S.; Boukherroub, R. Langmuir 2006, 22, 1660−1663. (34) Maalouli, N.; Barras, A.; Siriwardena, A.; Bouazaoui, M.; Boukherroub, R.; Szunerits, S. Analyst 2013, 138, 805−812. (35) Howarter, J. A.; Youngblood, J. P. Langmuir 2006, 22, 11142.

interesting alternative for LSPR based DNA sensing in an easy manner.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (J.S.); sabine. [email protected] (S. S.). Phone: +33 (0)1 44 27 55 12 (J.S.); +33 (0)3 62 53 17 25 (S. S.). Fax: +33 (0)1 44 27 60 33 (J.S.); +33 (0)3 62 53 17 01 (S. S.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The EU-FEDER and Interreg IV, project ‘‘Plasmobio’’, the Centre National de la Recherche Scientifique (CNRS), the Institut Universitaire de France (IUF), and the Nord-Pas-de Calais region are gratefully acknowledged for financial support.

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REFERENCES

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Approach for Plasmonic Based DNA Sensing: Amplification of the

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