Surface Plasmon-Enhanced Fluorescence Spectroscopy on Silver

Dec 8, 2010 - Physique de la Matière Condensée, Ecole Polytechnique, CNRS, 91128 Palaiseau, France, Department of Chemistry, University of Bath, Bat...
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Surface Plasmon-Enhanced Fluorescence Spectroscopy on Silver Based SPR Substrates Larbi Touahir,† A. Tobias A. Jenkins,‡ Rabah Boukherroub,§ Anne Chantal Gouget-Laemmel,† Jean-Noe¨l Chazalviel,† Jacques Peretti,† Franc¸ois Ozanam,*,† and Sabine Szunerits*,§ Physique de la Matie`re Condense´e, Ecole Polytechnique, CNRS, 91128 Palaiseau, France, Department of Chemistry, UniVersity of Bath, Bath BA2 7AY, AVon England, U.K., Institut de Recherche Interdisciplinaire (IRI, USR 3078), Parc de la Haute Borne, 50 AVenue de Halley, BP 70478, 59658 VilleneuVe d’Ascq, France, and Institut d’Electronique, de Microe´lectronique et de Nanotechnologie (IEMN, UMR 8520), Cite´ Scientifique, AVenue Poincare´, BP 60069, 59652 VilleneuVe d’Ascq, France ReceiVed: August 6, 2010; ReVised Manuscript ReceiVed: NoVember 9, 2010

For sensitive surface plasmon resonance (SPR) sensing the choice of the metal film and the strategy to bind the receptors to the SPR chip is critical. We have shown recently (Touahir, L.; Niedziolka-Jonsson, J.; Galpin, E.; Boukherroub, R.; Gouget-Laemmel, A. C.; Solomon, I.; Petukhov, M.; Chazalviel, J.-N.; Ozanam, F.; Szunerits, S. Langmuir 2010, 26, 6058) that a 5 nm thick layer of an amorphous silicon-carbon alloy (aSi1-xCx:H) deposited on a silver-based SPR interface can significantly enhance the sensitivity. In addition, the capping of a surface-plasmon active silver layer with a thin film of hydrogenated amorphous silicon-carbon alloy provides a practical solution for obtaining chemically stable SPR interfaces usable in conditions typical of bioassays with the additional advantage of benefiting from well-controlled processes for a robust covalent immobilization of biological probes to the interface. In this paper we demonstrate that the developed architecture in conjugation with an optimized surface functionalization scheme allows for a highly sensitive analysis of interfacial DNA-DNA binding interactions using surface plasmon-enhanced fluorescence (SPFS) as detection principle. The influence of the density of surface linked DNA probes on the recognition of 50 nM cDNA strands is presented. On an optimized surface (15% of acid-anchoring groups) DNA complementary probes with concentrations of 500 fM could be detected making this approach interesting compared to classical SPR experiments where nanomolar (nM) detection limits are conventionally reached. 1. Introduction Molecular analysis of nucleic acids has become an essential tool for applications in many different areas including biological research, clinical diagnostics, analysis of food, or environmental applications. Characteristic nucleic acid sequences can be used as distinctive markers for the identification and characterization of humans, animals, plants, prokaryotes, and viruses. The most commonly used technique representing the current standard of nucleic acid analysis is DNA microarrays.2,3 In a DNA microarray, an immobilized single stranded DNA (ss-DNA) probe hybridizes with a complementary sequence (known as target DNA) from solution. Thus, the interest in the development of immobilization strategies of DNA4-7 and the understanding of its hybridization at solid surfaces has increased considerably. On one hand, the efficiency and kinetics of the hybridization reaction in solution depend on factors such as ionic strength of the buffer, temperature, length of the nucleotide sequence, degree of mismatch, and G-C to A-T ratio.8 On the other hand, for the hybridization of target DNA in solution with surfaceconfined DNA, the heterogeneous rate constant is additionally influenced by factors such as steric accessibility, electrostatic repulsion, orientation constraints, or the rates of diffusion from * To whom correspondence should be addressed. E-mail: sabine.szunerits@ iri.univ-lille1.fr (S.S); franc¸[email protected] (F.O.). Phone: +33 3 62 53 17 25 (S.S.); +33 1 69 33 47 04 (F.O.). Fax: +33 3 62 53 17 10 (S.S.); +33 1 69 33 47 99 (F.O.). † Ecole Polytechnique. ‡ University of Bath. § Institut de Recherche Interdisciplinaire (IRI, USR 3078) and Institut d’Electronique, de Microe´lectronique et de Nanotechnologie (IEMN, UMR 8520).

the bulk solution to the interface.9 For addressing such complex issues, benefiting from investigation techniques sensitive enough to allow for monitoring in situ and in real time the hybridization at a solid surface is of prime interest. Surface plasmon resonance (SPR) is certainly the most widely used technique for in situ and real time measurements of probe-target interactions at surfaces.10-12 Gold surfaces are the substrate of choice for SPR measurements for mainly two reasons: gold is relatively stable in aqueous environments needed for monitoring bimolecular interactions and a versatile chemistry, mainly based on the formation of functional SAMs, has been developed and is well characterized. However, they are not the best candidates for achieving highly sensitive SPR sensing. Theoretical modeling of SPR in conducting metal oxide thin films has been performed by Franzen et al. who suggested that ITO could be a better suited substrate.13 However, this would require excitation and detection in the infrared range. In the conventional visible range, silver substrates appear to be the most appealing, because plasmon coupling exhibits a sharper angular resonance as compared to that on gold, yielding an increased sensitivity.14 However, silver suffers from a poor chemical stability which hampers its wide use for SPR sensing. There are mainly two strategies to circumvent this limitation. One is based on the use of bimetallic silver/gold layers.15,16 In this case, the usual thiol-on-gold chemistry can be used for coupling probes to the sensor surface. Alternatively, lamellar structures, where a thin dielectric film is deposited onto the surface plasmon active silver thin film protecting the underlying metal, were developed in the past few years.1,7,17-27 The used dielectrics are either oxide based,7,18-20,23,24,28 where the attach-

10.1021/jp107402r  2010 American Chemical Society Published on Web 12/08/2010

SPFS on Silver Based SPR Substrates ment of ligands to the surface is mainly achieved through silanization of surface hydroxyl groups, or based on the deposition of carbon26 and amorphous silicon-carbon alloys,1 which readily allows the modification with biomolecules of interest using well-developed and robust chemistry based on the attachment of alkene-containing molecules to the substrate through either UV light mediated formation of carbon-carbon bonds or Si-C bonds. Indeed, we have recently shown that amorphous silicon-carbon alloys (a-Si1-xCx:H) are interesting dielectric layers for efficiently protecting silver layers. They can be deposited as thin films, and changing the carbon content of the film allows for fine-tuning of the material properties.29,30 Increasing the carbon content allows for enlarging the optical band gap and decreasing the refractive index, which is beneficial for lamellar based SPR architectures.1 An amorphous siliconcarbon alloy with 37% carbon content (a-Si0.63C0.37:H) showed thus more favorable SPR characteristics than a film with 20% carbon content (a-Si0.80C0.20:H). Independent of the carbon content, the a-Si1-xCx:H capped structures proved to be chemically resistant, a mandatory requirement for biosensing applications, and convenient for a well-controlled immobilization of biological probes through covalent binding.1 Indeed, surface hydrogenated a-Si1-xCx:H can be conveniently functionalized by stable organic layers through robust Si-C covalent bonds in a similar way as crystalline silicon.31-35 The purpose of the present work is to examine whether similar advantages can be obtained by using a silver/a-Si1-xCx:H structure for designing sensitive and chemically stable surface plasmon-enhanced fluorescence spectroscopy (SPFS) substrates suited for in situ and real time studies of DNA hybridization at solid surfaces. Indeed, the technique of SPR suffers from a lack of sensitivity, which limits its use for the detection of low molecular-weight targets like DNA.36 In contrast, SPFS enables a gain in sensitivity by orders of magnitude,37-39 which makes the technique appealing in order to detect DNA hybridization processes. The excitation of surface-confined chromophores is achieved by the large enhancement of the evanescent electromagnetic field of the surface plasmon mode propagation along a metal/water interface.40 Since it was introduced in 1999,41 the merit of SPFS has been demonstrated in the studies of DNA duplexes for the detection of single mismatches42,43 and the detection of antibodies with attomolar sensitivity.44,45 In this contribution with the help of a mathematical model the fluorescence enhancement factors on different SPR interfaces will be determined. In addition, the influence of the density of the surface linked DNA probes on the hybridization kinetics and the final fluorescence intensity will be systematically studied. Finally, the possibility to discriminate between complementary and mismatched DNA will be looked at. We will show that a detection limit as in the upper femtomolar range can be achieved on an optimized SPFS interface. 2. Experimental Section 2.1. Materials. Hydrofluoric acid (HF) and acetic acid were purchased from Carlo Erba and were of VLSI grade. Undecylenic acid (99%) was supplied by Acros organics. NHydroxysuccinimide (NHS), N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC), phosphate buffer saline (10× PBS), sodium dodecyl sulfate (SDS), salmon sperm DNA, formamide, and saline-sodium citrate buffer (2× SCC) were obtained from Aldrich and used without further purification. Ultrapure water (Milli-Q, 18 MΩ cm) was used for the preparation of the solutions and for all rinses. Single stranded DNA was obtained from Invitrogen. The sequences chosen in this work were as follows: probe DNA

J. Phys. Chem. C, Vol. 114, No. 51, 2010 22583 (25mer), 5′-NH2-(CH2)6-[AAC-GCC-CAT-CTT-AAA-ATC-GACGCC-T], referred to as ON; target DNA, complementary PM, 5′-Cy5-AGG-CGT-CGA-TTT-TAA-GAT-GGG-CGT-T-3′; target DNA, four mismatches MM4, 5′Cy5-AGG-CGT-GCA-TTTTAA-GTA-GGG-CGT-T-3′; target DNA, one mismatch MM1, 5′Cy5-AGG-CGT-CGA-TTT-AAA-GAT-GGG-CGT-T-3′. 2.2. Preparation of the SPR Structures. Ti/Ag films were prepared by thermal evaporation of 5 nm of titanium and 38 nm of silver onto cleaned glass slides. Titanium is used as an adhesion layer between the glass surface and the silver layer. Amorphous silicon-carbon alloy layers were deposited onto Ti/Au and Ti/Ag films using plasma-enhanced chemical vapor deposition (PECVD) in a “low-power” regime.1,46,47 The following parameters were used: pressure ) 35 mTorr, temperature ) 250 °C, power density ) 0.06 W cm-2, and gas flow rate ) 20 cm3 min-1. The carbon content in the film has been found to be fully determined by the methane ratio in the gas mixture [CH4]/([SiH4]+[CH4]), as long as deposition remains performed in the low-power regime, which allows for adjusting the optical properties of the films. The correspondence between the carbon content x in the a-Si1-xCx:H film and the methane ratio has been determined by using a combination of various techniques including electron spectroscopies and elemental analysis.48 For the deposition of a thin film with the stoichiometry a-Si0.63C0.37: H, 94 at. % of [CH4] was used, while for an a-Si0.80C0.20:H film, 81 at. % is necessary. Heating the silver layer at 250 °C prior to a-Si0.63C0.37:H deposition might lead to oxidation of the silver film. Thus, on silver, a pretreatment with a hydrogen plasma (150 mT, 0.1 W/cm2) for 5 min has been applied to regenerate the silver surface just before turning on the silane/methane plasma for the deposition of the amorphous silicon-carbon films. 2.3. Monolayer Formation on Amorphous Silicon-Carbon Alloys. Acid-Terminated Surface. The surface of the Ti/Ag/ a-Si0.63C0.37:H structure was first etched with HF vapor for 15 s. The resulting hydrogen-terminated surface was placed at room temperature in a Schlenk tube containing previously deoxygenated neat undecylenic acid solution and irradiated at 312 nm for 3 h. The excess of unreacted and physisorbed reagent was removed by a final rinse in hot acetic acid for 30 min.33,49 Then, the sample was dried under nitrogen flow. NHS-Functionalized Surface. The conversion of the acid function to succinimidyl ester was accomplished as follows: the acid-terminated surface was covered with 10 mL of an aqueous solution of NHS (5 mM) and EDC (5 mM) and allowed to react for 90 min at 15 °C.15 The resulting surface was copiously rinsed with deionized water and dried under a stream of nitrogen. DNA Probe Immobilization. The NHS-terminated surface was reacted with amine-terminated oligonucleotide probes in PBS buffer (150 mM, 0.01% SDS pH 8.5) for 14-16 h.50 The unreacted ester groups were blocked with ethanolamine (50 mM, 15 min). The resulting surface was copiously rinsed with deionized water and dried under a stream of nitrogen. The probe concentration in the immobilization solution was chosen between 0.5 and 10 µM in order to affect the surface concentration of immobilized probes. In the case of experiments realized in a microarray format, the solution was deposited on the activated surface using a pin spotter (Biorobotics MicroGrid II). 2.4. DNA Hybridization. Hybridization Conditions Using Surface Plasmon-Enhanced Fluorescence (SPFS). DNA hybridization was performed by injection of 50 µL of complementary (100 fM to 50 nM) or noncDNA diluted in PBS (pH ) 7.4, 150 mM) with the help of a peristaltic pump at a speed

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of 1 µL/min. After the injection of 50 µL (corresponding to the volume where the solution had reached the SPR cell), the pump was stopped for 30 min to let hybridization occur, followed by a 5 min washing with PBS (pH ) 7.4, 150 mM). The regeneration of the hybridized interfaces were achieved using a mixture of formamide and 2.5× SSC [saline sodium citrate (50/50 by vol)],51 and the initial fluorescence signal was detected. 2.5. Instrumentation. Fluorescence Measurements. For end-point measurements, the fluorescence was measured with a scanner (Axon instrumentation personal 4100A). SPR and SPFS Measurements. The experimental setup used for SPR/SPFS measurements is directly inspired from that described by Knoll et al.8,44 The system consists of a He-Ne laser (Melles Griot Ltd., 5 mW, λ ) 632.8 nm) for exciting the surface plasmons, while two polarizers, positioned in the same alignment as the laser beam, adjust the intensity of the laser beam and cut off the transversal electric field polarization (only the transversal magnetic field has the adequate polarization to excite the surface plasmons). The sample holder and the photodiode are mounted on a two phase goniometer enabling angle dependent measurements. The goniometer is able to move 0.005° steps and is computer controlled. The sample holder is furthermore mounted onto two xy-tables and two tilting tables, which allow for the optimal adjustment of the setup. When the incident light reaches the base plane of the prism (Schott, LaSFN9, n ) 1.845), the light is reflected and focused by a lens (f ) 25 mm, Ovis), and the intensity is monitored by a photodiode located at 45° of the prism. The photodiode is connected to a lock-in amplifier to reduce the noise in the measurements. In order to measure fluorescence a photomultiplier tube (PMT) (Hamamatsu, Japan) is attached behind the sample holder and moves at the same rotation speed as the sample holder. The fluorescence is focused to the PMT by a collecting lens (f ) 50 mm, Ovis) positioned between the sample holder and the PMT and connected to a photon counter. A controlled shutter is integrated just after the laser, in order to prevent fluorophores from photobleaching. The whole setup was placed in a Faraday cage. A quartz flow cell, with a volume of 85 µL, is connected to a peristaltic pump (Rego Analog, Ismatec, U.K.). Real-time hybridization measurements of fluorescent labeled DNA is detected by monitoring the plasmon-enhanced fluorescence intensity recorded by the photomultiplier tube after adjusting the excitation incidence angle at a value about 1.5° lower than the resonance angle. After reaching a steady state, full angular spectra of reflectivity (for SPR) and fluorescence (for SPFS) are recorded. WinSpall 2.0 software (Max-Planck-Institute for Polymer Research, Mainz) was used to fit the experimental SPR curves with an optical model within the framework of the Maxwell macroscopic approach. 2.6. Calculations. The optical response of a layered structure was deduced, in the framework of the macroscopic Maxwell’s equations, from the resolution of the wave equation in each layer and from the continuity equations at the interfaces between two adjacent layers. In the case of linear isotropic media, s- and p-linearly polarized waves can be treated separately by a 2 × R ) and 2 matrix formalism,52 which yields the reflection (R1f R ) coefficients of the whole structure [i.e., the transmission (T1f complex amplitude of the reflected and transmitted electric field in the initial medium (labeled as 1) and the final medium (labeled as f) for a given (s- or p-) polarization (labeled R)]. Through the different media, the in-plane wavevector component

Touahir et al. q is conserved. In each media i, the wavevector component κi along the normal to the interface is obtained from

(nik0)2 ) q2 + κi2

(1)

where k0 ) 2π/λ is the wavevector modulus in vacuum and ni is the complex refractive index in medium i. At the interface between two adjacent media i and j, the reflection and transmission coefficients for s- and p-polarizations are given by

rijR )

aiR - ajR aiR + ajR

and tijR ) γijR

2aiR aiR + ajR

(2)

with asi ) κi/k0, γsij ) 1, api ) κi/n2i k0, γpij ) ni/nj. 3. Results and Discussion As shown recently by us, stable thin films of a-Si1-xCx:H with a carbon content between 20% and 37% can be deposited on SPR-active silver substrates.45 The best SPR signal in terms of resonance width and refractive index sensitivity was obtained on a 5 nm thick film with a carbon content of 37%. A sensitivity as high as 101 RIU-1 could be experimentally determined, being 2.8 times higher than that measured on simple gold-based SPR structures.1 To examine whether a similar advantage can be obtained for surface plasmon-enhanced fluorescence, we performed theoretical calculations of the enhancement factor in the presence and in the absence of a dielectric capping layer on top of the plasmon-active silver layer. For comparison, the simulations of angle dependent SPR reflectivity and fluorescence enhancement factors on naked and coated gold films are included. In addition, the influence of a multilayer structure, where the a-Si0.63C0.37:H dielectric overcoating is capped with a-Si0.80C0.20: H, is investigated. This investigation is based on the fact that higher carbon content means that less Si sites are available at the surface for chemical grafting, which could affect the probe immobilization performed on a molecular layer grafted on Si surface sites. Indeed, while using ATR FTIR spectroscopy, it has been shown that carboxydecyl groups are successfully transferred onto a-Si0.63C0.37:H;1,46 from fluorescence measurements it became evident (Figure 1A,B) that the immobilization of fluorescent oligonucleotides yields strong and homogeneous fluorescence signals on a-Si0.80C0.20:H, while on a-Si0.63C0.37:H the fluorescent signals are spatially inhomogeneous. The use of an a-Si0.80C0.20:H layer results indeed in better fluorescence signals due to favorable surface properties. In order to integrate the advantage of a a-Si0.80C0.20:H allowing more reliable and controlled probe immobilization, without influencing the refractive index SPR sensitivity (e.g., better sensitivity on a-Si0.63C0.37: H than on a-Si0.80C0.20:H), two different configurations, Ag/aSi0.63C0.37:H (3 nm)/a-Si0.80C0.20:H (2 nm) and Ag/a-Si0.63C0.37:H (3 nm)/a-Si0.80C0.20:H (3 nm), were formed experimentally. Figure 2C shows the resulting SPR spectra where the SPR signals of pure silver and silver coated with only 5 nm a-Si0.63C0.37:H is included for comparison. In both cases the plasmon resonance angle was shifted to higher angles, as compared to the Ag/a-Si0.63C0.37:H (5 nm) structure, while the resonance width fwhm increased only slightly. Referring to the classical figure of merit of SPR substrates obtained as the ratio ∆ΘSPR/fwhm (Figure 2D),53 the sensitivity decreases in both cases, but remains comparable to that of an uncoated silver film.

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Figure 1. Fluorescence images recorded on (A) a-Si0.80C0.20:H and (B) a-Si0.63C0.37:H after covalent linking of a fluorescent probe. (C) Scanning angle reflectivity curves of silver-based SPR substrates in water. Experimentally obtained values (b) were compared to theoretically calculated SPR curves (s) using WinSpall 2.0. (D) Change in the ratio ∆ΘSPR/fwhm with increasing refractive index n of the outer dielectric medium: silver only (black), silver + 5 nm a-Si0.63C0.37 (blue), silver + 3 nm a-Si0.63C0.37 + 2 nm a-Si0.80C0.20 (red), silver + 3 nm a-Si0.63C0.37 + 3 nm a-Si0.80C0.20 (green).

Figure 2. Simulation of the reflectivity and the squared intensity of the electrical field at the outer surface of several structures in the Kretschmann configuration using nprism ) 1.52. The squared electrical field intensity is referred to that of the incident field (enhancement factor). Simulated structures are (A) glass/Ti (5 nm)/gold (50 nm) (black), glass/Ti (5 nm)/silver (38 nm) (gray); (B) glass/Ti (5 nm)/silver (38 nm)/a-Si0.63C0.37 (5 nm) (gray) or glass/Ti (5 nm)/gold (50 nm)/a-Si0.63C0.37 (5 nm) (black); (C) glass/Ti (5 nm)/silver (38 nm)/a-Si0.80C0.20 (3 nm)/a-Si0.63C0.37 (3 nm) (gray) or glass/Ti (5 nm)/gold (50 nm)/a-Si0.80C0.20 (3 nm)/a-Si0.63C0.37 (3 nm) (black).

The sensitivity of the Ag/a-Si0.63C0.37:H (3 nm)/a-Si0.80C0.20:H (3 nm) structure was somewhat larger than that of the Ag/a-

Si0.63C0.37:H (3 nm)/a-Si0.80C0.20:H (2 nm) one, so that the former structure has been used in the following. The fluorescence-

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enhancement factor of the Ag/a-Si0.63C0.37:H (3 nm)/aSi0.80C0.20:H (3 nm) structure will be thus additionally computed in the following section. 3.1. Theoretical Determination of Fluorescence-Enhancement Factors on Multilayered Silver-Based SPR Interfaces. First, the case of a single noble metal layer m between two semi-infinite dielectric media 1 and f will be considered. The solution obtained from the 2 × 2 matrix formalism writes

RR1f )

TR1f )

R R 2iφm r1m + rmf e R R 2iφm 1 - rm1 rmfe R R iφm t1m + tmf e R R 2iφm 1 - rm1 rmfe

(3)

(4)

where φm ) κmz corresponds to the propagation into the metal slab along the z-direction normal to the interfaces. In ppolarization, when nf < n1, a peculiar situation occurs at the interface between the noble metal layer and the medium f of low refractive index when the metal slab is illuminated from medium 1 beyond the total reflection limit angle (that is when p and afp may take almost opposite q > nfk0). In this geometry, am p values since af becomes purely imaginary and increases from p 0 up to about (1/nf) [2(n1 - nf)/nf]1/2 with increasing q while am is of the order of 1/nm which is almost purely imaginary and p p and tmf are strongly negative. As a consequence, both rmf enhanced. The value corresponding to the excitation of the metal surface plasmon resonance yields a strong enhancement of the evanescent electromagnetic field at the metal surface (as given p ) and a narrow dip in the reflected field amplitude (as by T1f p p ). Since am is approximately equal to 1/nm, the given by R1f surface plasmon resonance (SPR) quality factor QSPR is given by

QSPR ) Im(nm)/Re(nm)

(5)

where nm is the (complex) refractive index of the metal. The largest field enhancement effects are obtained with the metal that exhibits the highest SPR quality factor at the optical frequency of interest. In the visible range, silver is the best candidate, while gold exhibits a SPR quality factor at least two times smaller than silver. For a multilayer structure, such as Ag/a-Si0.63C0.37:H (3 nm)/ p and T1fp can be a-Si0.80C0.20:H (3 nm), the solutions for R1f deduced in the framework of the 2 × 2 matrix formalism in the form of the generalization of eqs 3 and 4.54

RR1f )

R R R 2iφm R1m + Ψ1m1 Rmf e

TR1f )

R R 2iφm 1 - Rm1 Rmf e R R iφm T1m Tmfe R R 2iφm 1 - Rm1 Rmf e

(6)

(7)

In these expressions, the quantities RRij and TRij are no longer the reflection and transmission coefficients at the interface between two subsequent media but are the solution for the fields reflected and transmitted by the subset of layers stacked between media i and j, considering media i and j as semi-infinite. The

Figure 3. (A) Schematic representation of the used SPFS biosensor interface: DNA probes are covalently attached onto the a-Si0.8C0.2:H layer and available for hybridization with fluorescently labeled targets. (B) Influence of DNA probe density (a ) 0.5 µM, b ) 5 µM) on the fluorescence intensity recorded during hybridization with a 5 nM solution of (a, b) complementary and (c) noncDNA in PBS, recorded in a 5 nM solution, of PM targets with probes previously immobilized from a 5 µM solution (black, bottom) or a 500 nM solution (green, top). Notice the larger increase in the signal upon lowering the surface concentration of the probes. R R R R R quantity Ψ1m1 ) T1m Tm1 - R1m Rm1 should also appear in eq 3, but it is equal to unity at the interface between two subsequent media for which RRij and TRij must be replaced by rRij and tRij . From eqs 6 and 7, it is straightforward to obtain the solution for any multilayer structure by a simple recursive calculation. These expressions are very convenient for studying the effect of dielectric cap layers on the SPR. They indeed allow separating the multilayer structure into two subsets. The first one goes from p medium 1 to medium m. Its optical response, defined by R1m p and T1m , does not exhibit any plasmonic feature and remains unchanged when adding capping layers. The second subset goes from medium m to medium f. Its optical response defined, by p p and Tmf , exhibits SPR features and is modified by the Rmf additional cap layers. Such a separation is useful for assessing the SPR quality factor of the structure. Obviously the excitation conditions of the plasmon resonance are very sensitive to any change at the metal surface, but it is found that the field enhancement at the outer interface remains almost as strong as in the absence of a capping layer if the imaginary part of the refractive index and the thickness of the dielectric layers are small. Figure 2 shows the calculated variation of |Rp1f|2 and |Tp1f|2 as a function of the angle of incidence in a configuration where medium 1 is a glass substrate while the outer low refractive index medium is that of water. Figure 2A presents theoretically calculated reflectivity and fluorescence enhancement factors for naked silver (d ) 38 nm) and gold (d ) 42 nm) thin films when deposited on a glass substrate (n ) 1.52) and covered with a 5 nm titanium adhesion layer, while Figure 1B presents the results when both metals were coated with a 5-nm thick layer of

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Figure 4. (A) Schematic representation of the procedure to obtain diluted interfaces for DNA immobilization. (B) Influence of concentration of acid-anchoring groups on the change of fluorescence intensity during hybridization of 5 nM cDNA targets in PBS with DNA probes immobilized from a 0.5 µM solution. Concentration of anchoring groups in the molecular layer: 100% (green), 33% (red), 15% (blue), and 10% (black). (C) Change in Cy5 fluorescence intensity on a 15% anchoring group interfaces with complementary target concentrations between 100 fM and 5 nM and for 500 fM noncomplementary target.

a-Si0.63C0.37:H. On the gold-based SPR interfaces the fluorescence enhancement factor is decreased by about 30%, while on the Ag/a-Si0.63C0.37:H (5 nm) structure it remains twice as large as the one produced by an optimized bare gold layer. The computed fluorescence-enhancement factor for this Ag/aSi0.63C0.37:H (3 nm)/a-Si0.80C0.20:H (3 nm) structure (Figure 1C) is practically identical to that obtained for the Ag/a-Si0.63C0.37:H (5 nm) structure. In the following, a Ag/a-Si0.63C0.37:H (3 nm)/ a-Si0.80C0.20:H (3 nm) (Figure 3A) structure, having favorable surface chemistry (Figure 1), will be used for surface plasmon field-enhanced fluorescence spectroscopy (SPFS). 3.2. DNA-DNA Hybridization Detection Using Surface Plasmon Field-Enhanced Fluorescence Spectroscopy. Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) has been used on the multilayered SPR substrate (Figure 3A) for the real-time monitoring of interfacial binding between DNA probes immobilized at the substrate surface and targets of fluorescently labeled DNA strands. It is well-known that the efficiency and kinetics for the hybridization reaction target DNA in solution with surface-confined DNA is largely influenced by the steric accessibility and the rates of DNA diffusion from the bulk solution to the interface.9 The density of the probe DNA on the surface was first optimized. Figure 3B exhibits the SPFS signal after hybridization with 5 nM target DNA, when DNA probes have been immobilized from 5 and 0.5 µM solutions, respectively. In the case of the higher DNA surface concentration, the fluorescence signal reached a steady-state value quasiinstantaneously at the time scale of the measurements. In the case of a 10 times lower surface DNA density (Figure 3B) the time evolution of the SPFS signal is markedly different as the fluorescence signal increases progressively over time and reaches a steady state after ∼10 min. Moreover, the magnitude of the fluorescence signal at steady state is enhanced by a factor

of ∼6. This clearly indicates that high probe concentrations limit the hybridization efficiency and affect the hybridization kinetics. Moreover, the level of detected fluorescence was extremely low when hybridization was carried out with a noncDNA strand on a SPR interface where a probe DNA solution of 0.5 µM (e.g., low) was obtained. This indicates that the observed differences in fluorescence intensities are significant. In order to study the effect of probe density in a more controlled way, mixed molecular layers were grafted on the substrate by diluting the acid-anchoring groups necessary for DNA probe immobilization. This has been achieved by photochemical reaction in undecylenic acid in the presence of different concentrations of 1-decene, as sketched in Figure 4A. In this case, the ratio of surface-grafted carboxylic groups to surfacegrafted decyl groups has been determined from FT IR measurements.33 From Figure 4B one can see that decreasing the DNA surface concentration down to 15% results in a significant increase in the fluorescence signal using a 5 nM target DNA concentration and a 0.5 µM surface probe concentration to link the DNA strands, while on a 10% interface, the fluorescence signal is again lowered. A possible explanation of such an abrupt change could be sought in terms of changes in the average distance between immobilized DNA probes, which would become lower than a critical distance allowing for a free hybridization. In other words, it corresponds to a change from a situation where acid groups are mostly segregated in the mixed layer after grafting as suggested by Aureau et al.,55 to a situation where they are more randomly dispersed. From a practical viewpoint, the substrates obtained by grafting a 15%-acid molecular layer are also those exhibiting the best sensitivity. As shown in Figure 4C, after immobilization of DNA from a 0.5 µM solution, they allow detecting complementary solution DNA strands below 500 fM. The fluorescence signal obtained

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Figure 5. Hybridization kinetics of various targets at 5 nM concentration [PM (top, green), MM1 (middle, red), MM4 (bottom, blue)] with the probes immobilized from a 500 nM solution on a 15% acid a-Si0.8C0.2:H surface.

with a 100 fM cDNA solution is indistinguishable from a noncDNA solution of 500 fM, thus defining the detection limit. This is lower than the reported detection limit of 200 pM for DNA-PNA56 or DNA hybridization on phosphorus dendrimer multilayer films57 using SPFS. Finally, the Ag/a-Si0.63C0.37:H (3 nm)/a-Si0.80C0.20:H (3 nm) interfaces with 15%-acid molecular layer were tested for their ability to distinguish between perfectly matched or slightly mismatched target (four and one) sequences. At a target concentration of 5 nM, the fluorescence signal sets in instantaneously and provides a clear distinction between perfectly matched targets and ones with four and one mismatches (Figure 5). 4. Conclusion We have demonstrated that capping a silver layer with a thin layer of hydrogenated amorphous silicon-carbon alloy provides a practical solution for obtaining efficient SPFS substrates. With this architecture, the silver layer is efficiently protected against corrosion, which allows for obtaining a stable sensor usable in conditions typical of bioassays. Probe DNA crowding at the surface is a major factor affecting the hybridization efficiency. Surfaces with only 15% acid-anchoring groups proved to be optimal for sensitive SPFS sensing. On such interfaces, the resulting limit of detection (LOD ≈ 500 fM) overcomes that of gold-based SPFS sensors, with the extra advantage of benefiting from well-controlled processes for a robust covalent immobilization of biological probes on the substrate. A very good discrimination between perfectly matched and single mismatched sequences can be obtained on these interfaces. Acknowledgment. Financial support from Fondation de l’E´cole Polytechnique, Conseil Ge´ne´ral de l’Essonne (Project SiBioslides), the EU-FEDER and Interreg IV (Project “Plasmobio”), the Centre National de la Recherche Scientifique (CNRS), and the Nord Pas-de-Calais region is gratefully acknowledged. References and Notes (1) Touahir, L.; Niedziolka-Jonsson, J.; Galopin, E.; Boukherroub, R.; Gouget-Laemmel, A. C.; Solomon, I.; Petukhov, M.; Chazalviel, J.-N.; Ozanam, F.; Szunerits, S. Langmuir 2010, 26, 6058.

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