DEVICE "

TECHNICAL FIELD

The present invention concerns a method for the preparation of a substrate that can be used in systems for performing molecular analyses, such as Surface Enhanced Raman Spectroscopy (SERS), fluorescence, luminescence and plasmonic detection .

BACKGROUND ART

With particular reference to SERS, Raman spectroscopy is a spectroscopy technique used in the physics and chemistry of condensed matter to study, for example, the vibrational behaviour of molecular systems.

In Raman spectroscopy experiments, a beam of approximately monochromatic light in a particular wavelength range passes through a sample of a molecule which emits a diffuse light spectrum.

The spectrum of the wavelengths emitted by the molecule is called "Raman spectrum" and the light emitted is called "Raman diffuse light". A Raman spectrum can detect the vibrational or electronic energy levels of a molecule. Different molecules produce different Raman spectra which can be used to identify the molecules or also to determine the structure of a molecule .

Raman spectroscopy is used to study the transitions between molecular energy states when the photons interact with the molecules, thanks to a shifting of the energy of the diffuse photons .

The Raman diffusion of a molecule consists of two processes. The molecule, which is in a certain energy state, is first excited by incident photons in a different energy state (both real and virtual), which is generally in the optical frequency domain. The excited molecule then emits light at a frequency which may be lower (for example Stokes diffusion) or higher (for example anti-Stokes diffusion) than that of the excitation photons.

The Raman spectrum of different molecules or species can have characteristic peaks which can be used to identify the species .

Raman spectroscopy is therefore a useful technique in a wide variety of applications for detection and identification of chemical and/or biological compounds.

However, the Raman diffusion process is very inefficient and, as a result, methods are currently being searched for in order to improve the process. The Raman light generated by molecules or species adsorbed on surfaces of a few nanometres of a structured metal support can be 10 ³ -10 ⁴ times greater than the Raman light generated by the same species in solution or in the gaseous phase. This amplification process is called Surface Enhanced Raman Spectroscopy (SERS) . In recent years SERS has become established as a routine and very powerful tool for the study of molecular structures, for the characterisation of interface systems and thin film systems and for the detection of single molecules.

The biomedical field has seen great technical advances from the development of SERS devices which make use of metal nanoparticles .

Numerous techniques are known for the production of metal nanostructure matrixes for SERS applications, for example lithographic techniques, like electronic beam lithography ^1-3 and laser processing ^4-8 .

Metal nanoparticles are typically obtained on a substrate by heating a gold film previously deposited on the substrate by laser irradiation ^4,5,8 .

Other researchers have used an approach by interference between two rays to form a drawing on a photosensitive chalcogenide glass film followed by coating of the nanostructured surface with thin film ⁶ .

Substrates of silicon in aqueous solution of silver nitrate were processed with a femtosecond laser to simultaneously obtain the generation of nanostructure networks on the surface of the substrate and the formation of silver nanoparticles on the surface of the nanostructures themselves ⁷ .

Other methods of preparation of SERS devices entail the deposition from solvent of chemically synthesized colloidal particles ⁹ . Other groups have studied self-assembly of gold particles on glass surfaces chemically functionalized with organosilanes ¹⁰ .

Many of these techniques have numerous limitations, for example high costs, long preparation times, toxicity and the fact that they allow the treatment of small areas.

The need is therefore felt in the art for a method for the preparation of a substrate for a plasmonic device which is rapid, inexpensive, does not require the use of toxic materials (chemically synthesized colloidal nanoparticles) and which has improved detection sensitivity.

DISCLOSURE OF INVENTION

The object of the present invention is therefore to provide a new method for the preparation of a substrate for a plasmonic device which is free from the drawbacks of the methods described above.

Said object is achieved by the present invention, since it relates to a method according to claim 1.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described in detail with reference to the Figures of the accompanying drawings, in which :

- Figure 1 schematically illustrates the steps of the method according to the invention;

- Figure 2 schematically illustrates a pointed microstructure functionalized with ( 3-mercaptopropyl ) -trimethoxysilane (panel a) and the anchoring of a gold nanoparticle on the same (panel b) ;

- Figure 3 illustrates SEM images of a pointed microstructure matrix at two different enlargements (panel a: X25, panel b: X400) ;

Figure 4 illustrates a TEM image of gold nanoparticles obtained by laser ablation of a gold sheet in deionized water and a histogram relative to the dimensional distribution of the same;

Figure 5 illustrates the Raman spectrum of the gold nanoparticles obtained by laser ablation of a gold sheet in deionized water (a) and obtained chemically (b) ;

Figure 6 shows the images obtained under the optical microscope of a pointed microstructure with gold nanoparticles supported on the point (panel a) and on the base (panel b) of the microstructure, a schematic representation of the pointed microstructure after the deposition of gold nanoparticles (panel c) and the relative Raman spectrum of the point (panel d) ;

- Figure 7 illustrates the SERS spectra of the Cresyl Violet (CV) obtained using respectively a matrix of pointed microstructures before the deposition of gold nanoparticles (panel a) , plane silicon after deposition of a thin layer of gold (lOnm) (panel b) , a pointed microstructure matrix after deposition of a thin layer of gold (10 nm) using the evaporation technique (panel c) and a pointed microstructure matrix after deposition of gold nanoparticles (panel d) ;

- Figure 8 illustrates the intensity of the band at 592 cm-1, 674 cm-1 and 1184 cm-1 of the SERS spectra of the CV obtained on different substrates using respectively a pointed microstructure matrix before the deposition of gold nanoparticles (sample A), plane silicon after deposition of a thin layer of gold (sample B) , a pointed microstructure matrix after deposition of a thin layer of gold (sample C) and a pointed microstructure matrix after deposition of gold nanoparticles (sample D) .

BEST MODE FOR CARRYING OUT THE INVENTION

According to a first aspect of the invention, a method is provided for the preparation of a substrate for a plasmonic device. With reference to Figure 1, according to the method of the invention, a first surface 2 of a substrate 1, made of photosensitive glass, is microstructured by selective exposure to the light.

By "photosensitive glass" we mean totally transparent glass belonging to the family of lithium silicate glass composed of approximately 70% S1O ₂ , 30% B ₂ O ₃ .

In one embodiment said microstructuring can be achieved by treating the surface 2 with a light beam. Said treatment can be performed by laser ablation.

In one embodiment, said treatment can be performed by irradiation with a laser or UV lamp, along a pre-defined path, for example defined by masking the surface 2 using a masking element 3, for example a photolithographic template or a stencil template. Said masking element 3 can be positioned near the surface 2 or used to obtain an image on the surface 2 by means of an appropriate optical protection system. The selective irradiation of the surface 2 described above has a geometry such as to define on the same, in at least one direction, a first plurality of irradiated portions 5 and a second plurality of non-irradiated portions 4.

After the light irradiation and removal of the masking element 3, the substrate 1 is heated.

In particular the heating can be conducted with a temperature gradient of 2-8°C/min from ambient temperature to 250-750°C; the substrate is then heated at a speed of l-5°C/min from 250- 750°C to 355-855°C and lastly said temperature is maintained at 355-855°C for 1 hour.

After the heating, the substrate 1 is treated with a strong acid, for example hydrofluoric acid. The acid, in contact with the second plurality of unmasked portions 5 treated by UV irradiation, determines an erosion at a speed higher than that at which the first plurality of masked portions 4 is eroded. In this way a substrate 1 is obtained having a plurality of substantially conical microstructures 6.

On said plurality of microstructures 6 a layer of metal nanoparticles 7 is then deposited, preferably gold, silver, copper and their alloys.

The metal nanoparticles 7 are obtained by means of laser ablation of a metal sheet in water or in an aqueous solution (for example aqueous solution of oxygenated water or ethanol) . Preferably the metal sheet is made of gold, silver, copper and their alloys. In particular the metal nanoparticles 7 are prepared as described in Intartaglia et al. (Phys. Chem. Chem. Phys., 2013, 15, 3075, DOI : 10.1039/C2CP42656K) .

Advantageously, the production of metal nanoparticles 7 by means of laser ablation in water or in an aqueous solution allows nanoparticles to be obtained free from organic residues due to the use of solvents. Compared to the use of metal nanoparticles obtained according to traditional techniques, said metal nanoparticles 7, once deposited on the plurality of microstructures 6, do not generate a background noise in transmission of the signal due to the presence of organic residues on their surface and surprisingly determine a considerable increase in the intensity of the signal detected.

To promote the adhesion of the metal nanoparticles 7 to the plurality of microstructures 6, the latter can be functionalized with a functionalizing agent, in particular an organosilane with a functional group chosen in the group consisting of -CN, -N¾ and -SH, preferably the organosilane is selected in the group consisting of ( 3-mercaptopropyl ) - trimethoxysilane, (3-mercaptopropyl) -methyl-dimethoxysilane .

Further characteristics of the present invention will become clear from the following description of some merely illustrative and non-limiting examples.

The following abbreviations are used in the examples below: fs (femtoseconds), ns (nanoseconds), min (minutes), h (hours), g (grams), ml (millilitres ) , Dm (micrometres), nm (nanometres), cm (centimetres), vol% (percentage in volume), Da (Dalton) , mmol (millimoles ) , M (molar), W (Watt), kHz (kilohertz), mJ (milli oule ) , °C (degrees centigrade), %vol (percentage in volume), KrF (krypton fluoride), MPTMS ( 3-mercaptopropyl- trimethoxysilane) , NH40H (ammonium hydroxide), H ₂ O ₂ (oxygenated water) , ¾0 (water) , HC1 (hydrochloric acid) , NPs (nanoparticles), Au (gold), -SH (thiolic functionality of the MPTMS), Au-S (gold-sulphur bond), CV (Cresyl Violet, hexamethyl pararosaniline chloride), SEM (scanning electron microscope) , TEM (transmission electron microscope) . Example 1

Preparation of a plasmonic device

The method according to the invention is performed in three steps. The first step entails irradiation with a UV laser of a biocompatible silica substrate covered by a masking element, followed by a heating step and by chemical treatment with hydrofluoric acid in an ultrasound bath.

In particular, a Foturan ^® glass support is selectively irradiated by projection of the image of a masking element consisting of chromium (absorbent) on quartz (transparent) which has a honeycomb pattern.

The unmasked portion of the glass is treated with KrF excimer laser with one single pulse at 248 nm with duration of 20 ns and fluence of 0.1 J" cnf ² .

The masking element is then removed and the photosensitive glass support is heated in a programmable oven to obtain the formation of a crystalline phase of lithium methasilicate, in which the temperature is first set with a temperature gradient from ambient temperature to 500°C at 5°C/min, then said temperature is maintained constant for 1 h and then further increased to 605°C at a speed of 3°C/min and maintained at said temperature for another hour.

The substrate is then immersed in an aqueous solution of hydrofluoric acid at 10 vol% in an ultrasound bath to rapidly remove the area treated with the laser and obtain a pointed microstructure matrix. After 2 minutes from immersion in the aqueous solution of hydrofluoric acid, etching of the surface portions exposed to the laser treatment begins, then extending also towards the non-exposed portions. The formation of the conical structures is the consequence both of the different erosion speed at different depths of the substrate and of the different erosion speed of the portions of substrate sensitized with the laser treatment and those not exposed; the surface glass is exposed to a longer erosion time than the internal regions, giving rise to a conical formation of the areas exposed to the laser. Once the substrate is immersed in the aqueous solution of hydrofluoric acid, the erosion speed is greater for the portions treated with the laser and slower on the non-exposed portions. In a lateral transverse section of the support 1, the eroded volumes take on a substantially trapezoidal shape (Figure 1 panel b) ; as the erosion proceeds, the adjacent trapezoidal volumes grow until they touch, forming a pointed microstructure matrix (Figure 1 panel c) .

The surface of the microstructure matrix is subsequently functionalized by treatment with ( 3-mercaptopropyl ) - trimethoxysilane (MPTMS) as follows. The microstructure matrix is washed and rinsed with distilled water and subject to sonication in ethanol for 2 minutes before being cleaned with a solution of ammonium hydroxide, oxygenated water at 30 vol% and water NH ₄ OH : H ₂ O ₂ : ¾0 1:1:5 in volume at 80°C for 5 minutes and subsequently with a solution of hydrochloric acid, oxygenated water at 30 vol% and water HC1 : H ₂ O ₂ : ¾0 1:1:5 5 in volume at 80°C for 5 minutes. Subsequently the microstructure matrix is rinsed three times with distilled water and left to dry. A solution of MPTMS in toluene is prepared by addition of 2 ml of MPTMS at 20 ml of toluene. The microstructure matrix is then immersed in the solution of the MPTMS in toluene at ambient temperature (10 %vol) and incubated for 1 h in order to surface-functionalize it with MPTMS. The functionalized matrix is then washed three times with toluene to remove the non-bound MPTMS, left to dry and conserved in a dry nitrogen atmosphere .

The synthesis of metal nanoparticles (NPs) free from organic solvent is performed by laser ablation, with a femtosecond laser, in deionized water. The laser used is a Ti-sapphire laser with amplitude pulse of 110 fs and centred at 800 nm operating at a repetition frequency of 1 kHz. The laser beam is focused by means of a lens with focal distance of 10 cm on a gold sheet (purity 99.999%, Alfa Aesar GmbH & Co Karlsruhe, Germany) positioned on the bottom of a quartz cuvette containing 1 ml of deionized water. Before irradiation the sheet is polished and washed several times with deionized water in order to remove the impurities from the surface of the sheet. During the laser ablation process the sheet is moved by a rotation system (T-cube DC servo controller, Thor Tabs) to obtain a uniform irradiation of the same and an aqueous dispersion of metal nanoparticles of gold.

The microstructure matrix functionalized with MPTMS is immersed in the dispersion containing the metal nanoparticles of gold, and incubated for 1 h at ambient temperature to allow the formation of a bond (Au-S) between the metal nanoparticles of Au and the -SH group of the MPTMS anchored to the surface of the matrix (Figure 2) . Subsequently the microstructure matrix bearing the metal nanoparticles of gold is washed three times with distilled water and left to dry in nitrogen atmosphere .

Example 2

Characterisation of the substrate with plurality of microstructures

The substrate obtained in the preceding example was characterised before surface functionalization and subsequent deposition of the metal nanoparticles.

Figure 3 shows SEM images of a pointed microstructure matrix, obtained by using a masking element consisting of chromium on quartz with a honeycomb pattern, at two different enlargements (panel a: X25, panel b: X400) . The SEM analyses show the typical three-dimensional structure of the structured glass surface after the laser treatment and the chemical surface erosion. The periodic microstructures cover an area of the glass surface of 5 mm diameter but the microstructuring process can be applied to obtain larger surfaces by appropriate optimisation of the laser parameters. The microstructures show a periodicity of 75 Dm and a form ratio of 1:1. The form ratio of the microstructures can furthermore be modified by varying the chemical surface erosion time or the concentration of acid.

Example 3

Characterisation of the gold nanoparticles

The gold nanoparticles obtained in example 1 were characterised prior to deposition of the same on the plurality of microstructures by the use of an electron transmission microscope (TEM) JEOL Jem 1011 operating with an acceleration voltage of 100 keV.

A drop of suspension of the gold nanoparticles in water was deposited directly on a copper grille with 300 mesh covered with carbon and the water was left to evaporate.

Figure 4 shows the TEM images of the nanoparticles obtained by laser ablation with a femtosecond laser of a gold sheet in deionized water. From the images, isolated gold nanoparticles with a substantially spherical morphology and smooth surface can be seen. Figure 4 also shows the histogram relative to the dimensional distribution of the gold nanoparticles, obtained from evaluation of the dimensions of more than 300 particles and showing a mean particle diameter dimension of 8 nm.

The nanoparticles were furthermore characterised by Raman spectroscopy; the spectrum obtained (Figure 5) shows the complete absence of bands in the spectrum range analysed, demonstrating the high purity of the gold nanoparticles obtained .

Example 4 Characterisation of the substrate after deposition of metal nanoparticles of gold

The optical characterisation of the microstructure matrix after deposition of metal nanoparticles of gold was performed by optical microscope and Raman spectroscopy. The optical analysis was performed by using a Leica DM2500M optical microscope equipped with a DFC290 camera and 100X lens. As shown in Figure 6 panels a and b, the optical analysis showed the presence of the nanoparticles throughout the surface of the microstructure matrix (Figure 6 panel c) .

The bond between nanoparticles and microstructure was determined by Raman spectroscopy via the use of a Raman Renishaw inVia microscope with 150X lens (NA - 0.95) at ambient temperature. The spectrum was recorded in backscattering geometry irradiating at a wavelength of 633 nm with a power of 55 mW and acquisition time of 0.1 seconds. By using a grid with 1800 lines/mm, a spectrum resolution of approximately 1.1 cm was obtained ^-1 . The Raman spectrum of a pointed microstructure after deposition of gold nanoparticles (Figure 6 panel d) shows a transition around 253 cnf ¹ corresponding to the vibrational mode of the Au-S group which confirms the presence of metal nanoparticles of gold on the surface of the matrix.

Example 5

Comparison with other substrates for SERS

The high detectability limit is highlighted by comparison with a microstructure matrix without nanoparticles and with a microstructure matrix on which a thin film of 10 nm of gold was deposited physically by evaporation. The measurement was taken using Cresyl Violet (CV) as the analyte. The CV was deposited from aqueous solution (1 nM) on the different types of substrate. Figure 7 shows the SERS spectrums of the CV obtained using a pointed microstructure matrix without nanoparticles (panel a) , plane silicon with a thin layer of gold on top (panel b) , a pointed microstructure matrix with a thin layer of gold (panel c) and a pointed microstructure matrix with gold nanoparticles obtained according to the method of the invention (panel d) . The Raman measurements show that the spectrum recorded in the case of the microstructure matrix without gold (panel a of Figure 7) does not have absorption bands; these can be seen, on the other hand, when the microstructure matrix is covered by a thin layer of gold

(panel c of Figure 7) and when the flat substrate is covered by a thin layer of gold (panel b of Figure 7) . The use of a microstructure matrix with gold nanoparticles (panel d of Figure 7) allows a spectrum to be obtained with numerous and intense peaks characteristic of the CV. In particular a very intense peak is recorded at 590 cm-1, and other less intense peaks between 200 and 1200 cm-1. The intensity of this signal can be attributed to the proximity between the aminic group of the CV and the surface of the gold, which entails an increase in Raman scattering. The intensity of the band at 592 cm-1 for the four samples is reported in Figure 8. The amplification factor in the case of use of the pointed microstructure matrix with gold nanoparticles (sample D) is 2.5xl0 ⁵ with respect to the use of plane silicon with a thin layer of gold on top

(sample B) .

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