Anne Chantal Gouget-Laemmel

Localized surface plasmon-enhanced fluorescence spectroscopy for highly-sensitive real-time detection of DNA hybridization

Versatile and highly-sensitive detection of DNA hybridization is described using metal nanostructures-enhanced fluorescence (MEF) emission intensity when fluorescently-labeled DNA oligomers are covalently immobilized on a nanometer-thin amorphous silicon-carbon layer capping the metal nanostructures. The MEF structures are formed by thermal deposition of silver, gold or silver/gold thin films on glass surfaces and post-annealing at 500 degrees C. The choice of the metal film allows for tuning the optical properties of the interface. The metallic nanostructures are subsequently coated with an amorphous thin silicon-carbon alloy (a-Si(0.80)C(0.20): H) layer deposited by PECVD. Carboxydecyl groups are attached on these surfaces through hydrosilylation then reacted with amine-terminated single-stranded DNA oligomers, forming a covalent link. The immobilized DNA is hybridized with its complementary strand carrying a fluorescent label. Through optimization of the thickness of the a-Si(0.80)C(0.20): H alloy overlayer and by working close to resonance conditions for plasmon and fluorophore excitation, the hybridization of very dilute oligomers (5 fM) is easily detected, and the hybridization kinetics can be monitored in situ and in real-time.

Surface Plasmon Resonance on Gold and Silver Films Coated with Thin Layers of Amorphous Silicon−Carbon Alloys

The paper reports on a novel surface plasmon resonance (SPR) substrate architecture based on the coating of a gold (Au) or silver (Ag) substrate with 5 nm thin amorphous silicon-carbon alloy films. Ag/a-Si(1-x)C(x):H and Au/a-Si(1-x)C(x):H multilayers are found to provide a significant advantage in terms of sensitivity over both Ag and Au for SPR refractive index sensing. The possibility for the subsequent linking of stable organic monolayers through Si-C bonds is demonstrated. In a proof-of-principle experiment that this structure can be used for real-time biosensing experiments, amine terminated biotin was covalently linked to the acid-terminated SPR surface and the specific streptavidin-biotin interaction recorded.

Molecular monolayers on silicon as substrates for biosensors

(111) silicon surfaces can be controlled down to atomic level and offer a remarkable starting point for elaborating nanostructures. Hydrogenated surfaces are obtained by oxide dissolution in hydrofluoric acid or ammonium fluoride solution. Organic species are grafted onto the hydrogenated surface by a hydrosilylation reaction, providing a robust covalent Si-C bonding. Finally, probe molecules can be anchored to the organic end group, paving the way to the elaboration of sensors. Fluorescence detection is hampered by the high refractive index of silicon. However, improved sensitivity is obtained by replacing the bulk silicon substrate by a thin layer of amorphous silicon deposited on a reflector. The development of a novel hybrid SPR interface by the deposition of an amorphous silicon-carbon alloy is also presented. Such an interface allows the subsequent linking of stable organic monolayers through Si-C bonds for a plasmonic detection. On the other hand, the semiconducting properties of silicon can be used to implement field-effect label-free detection. However, the electrostatic interaction between adsorbed species may lead to a spreading of the adsorption isotherms, which should not be overlooked in practical operating conditions of the sensor. Atomically flat silicon surfaces may allow for measuring recognition interactions with local-probe microscopy.

Development of a metal-chelated plasmonic interface for the linking of His-peptides with a droplet-based surface plasmon resonance read-off scheme.

Monolayers of metal complexes were covalently attached to the surface of lamellar SPR interfaces (Ti/Ag/a-Si(0.63)C(0.37)) for binding histidine-tagged peptides with a controlled molecular orientation. The method is based on the activation of surface acid groups with N-hydroxysuccinimide (NHS), followed by an amidation reaction with (S)-N-(5-amino-1-carboxypentyl)iminodiacetic acid (NTA). FTIR and X-ray photoelectron spectroscopy (XPS) were used to characterize each surface modification step. The NTA modified SPR interface effectively chelated Cu(2+) ions. Once loaded with metal ions, the modified SPR interface was able to bind specifically to histidine-tagged peptides. The binding process was followed by surface plasmon resonance (SPR) in a droplet based configuration. The Cu(2+)-NTA modified interface showed protein loading comparable to commercially available NTA chips based on dextran chemistry and can thus be regarded as an interesting alternative. The sensor interface can be reused several times due to the easy regeneration step using ethylenediaminetetraacetic acid (EDTA) treatment.

Amorphous silicon-carbon alloys for efficient localized surface plasmon resonance sensing.

This paper describes a novel platform for preparing localized surface plasmon resonance (LSPR) sensing surfaces. It is based on the coating of gold nanostructures deposited on glass with an amorphous silicon-carbon alloy overcoating. The interest in coating the Au NSs with an amorphous silicon-carbon alloy resides in the possibility of incorporating carboxyl functions directly onto the surface via Si-C covalent bonds. This permits the use of hyrdosilylation reactions to modify the sensor surface. The use of this multilayer structure for the detection of hybridization events is discussed.

Influence of the Molecular Design on the Antifouling Performance of Poly(ethylene glycol) Monolayers Grafted on (111) Si

Various poly(ethylene glycol) monomethyl ether moieties were grafted onto hydrogenated silicon surfaces in order to investigate the influence of the molecular design on the antifouling performance of such coatings. The grafted chains were either oligo(ethylene oxide) chains (EG)(n)OMe bound to silicon via Si-O-C covalent bonds, or hybrid alkyl/oligo(ethylene oxide) chains C(p)(EG)(n)OMe bound via Si-C covalent bonds (from home-synthesized precursors). Quantitative IR spectroscopy gave the molecular coverage of the grafted layers, and AFM imaging demonstrated that a proper surfactinated rinse yields C(p)(EG)(n)OMe layers free of unwanted residues. The protein-repellent character of these grafted layers (here, toward BSA) was studied by IR and AFM imaging. C(p)(EG)(n)OMe layers exhibit a lower surface concentration than (EG)(n)OMe layers, because of the presence of a solvent in the grafting solution; they however demonstrate high resistance against BSA adsorption for high values of the n/p ratio and a higher stability than (EG)(n)OMe. This behavior is consistently explained by the poor ordering capability of the alkyl part of the layer, contrary to what is observed for similar layers on Au, and the key role of an entangled arrangement of the ethylene oxide chains which forms when these chains are long enough.

Plasmonic properties of silver nanostructures coated with an amorphous silicon–carbon alloy and their applications for sensitive sensing of DNA hybridization

The use of an amorphous silicon-carbon alloy overcoating on silver nanostructures in a localized surface plasmon resonance (LSPR) sensing platform allows for decreasing the detection limit by an order of magnitude as compared to sensors based on gold nanostructures deposited on glass. In addition, silver based multilayer structures show a distinct plasmonic behaviour as compared to gold based nanostructures, which provides the sensor with an increased short-range sensitivity and a decreased long-range sensitivity.

Highly sensitive and reusable fluorescence microarrays based on hydrogenated amorphous silicon-carbon alloys

We have designed a new architecture of fluorescent microarrays based on a thin layer of hydrogenated amorphous silicon-carbon alloy (a-Si(0.85)C(0.15):H) deposited on an aluminium-on-glass back reflector. These substrates are modified with an organic monolayer anchored through Si-C bonds and terminated with carboxyl groups, allowing for the covalent immobilization of biological probes. The fluorescence yield is maximized by optimization of the a-Si(0.85)C(0.15):H layer thickness. This approach is assessed for DNA recognition, demonstrating an increase in sensitivity by over one order of magnitude as compared to commercial slides, and the possibility of following in situ the molecular recognition event (hybridization). The immobilization chemistry provides these substrates with a superior chemical stability toward ageing or long-term exposure to physiological buffers, which allows for many successive hybridization/dehybridization cycles without measurable changes in performance.

Etching and Chemical Control of the Silicon Nitride Surface

Silicon nitride is used for many technological applications, but a quantitative knowledge of its surface chemistry is still lacking. Native oxynitride at the surface is generally removed using fluorinated etchants, but the chemical composition of surfaces still needs to be determined. In this work, the thinning (etching efficiency) of the layers after treatments in HF and NH4F solutions has been followed by using spectroscopic ellipsometry. A quantitative estimation of the chemical bonds found on the surface is obtained by a combination of infrared absorption spectroscopy in ATR mode, X-ray photoelectron spectroscopy, and colorimetry. Si-F bonds are the majority species present at the surface after silicon nitride etching; some Si-OH and a few Si-NHx bonds are also present. No Si-H bonds are present, an unfavorable feature for surface functionalization in view of the interest of such mildly reactive groups for achieving stable covalent grafting. Mechanisms are described to support the experimental results, and two methods are proposed for generating surface SiH species: enriching the material in silicon, or submitting the etched surface to a H2 plasma treatment.

Voltammetric Behavior of Peptide-Modified Porous Silicon after Metal Complexation

Hybrid nanomaterials based on organic layer covalently grafted on porous silicon (PSi) nanostructure appear as promising systems for innovative applications such as detecting of traces amounts and/or removing metal cations in water effluents. In this work, we focused on the functionalization of the PSi nanostructure by the peptide GlyCysGlyCys, which forms stable complexes with metal ions. This property is exploited to achieve toxic metal recognition in water using electrochemical methods. Peptide immobilization was achieved using multi-step reactions; GlyCysGlyCys was anchored on a previously prepared carboxyl-terminated PSi surface, using EDC/NHS coupling agents. This scheme is compatible with the mild conditions required for preserving the probe activity of the peptide. At each step of the functionalization, the surface was monitored by Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). Electrochemical behavior of such modified electrode was carried out after Nickel accumulation on the surface, by means of cyclic voltammetry. The recorded cyclic voltammograms showed a quasi-irreversible process corresponding to the Ni2+/Ni0 couple.