WO2022136262A1

WO2022136262A1 - Real time trace detection

Info

Publication number: WO2022136262A1
Application number: PCT/EP2021/086772
Authority: WO
Inventors: Javier PLOU IZQUIERDO; Luis Manuel LIZ-MARZÁN; Isabel García Martín; Mathias CHARCONNET
Original assignee: ASOCIACIÓN CENTRO DE INVESTIGACIÓN COOPERATIVA EN BIOMATERIALES - CIC biomaGUNE; ASOCIACIÓN CENTRO DE INVESTIGACIÓN COOPERATIVA EN NANOCIENCIAS "CIC nanoGUNE"; Consorcio Centro de Investigación Biomédica en Red, M.P.
Priority date: 2020-12-21
Filing date: 2021-12-20
Publication date: 2022-06-30

Abstract

The present invention provides a layered substrate comprising (a) an electromagnetically active layer, (b) a support layer adjacent to the electromagnetically active layer, and (c) a thermolabile sheathing layer adjacent to the electromagnetically active layer, wherein: at least one of the layers (b) or (c) adjacent to the electromagnetically active layer is transparent to an incident electromagnetic radiation of wavelength W; the sheathing layer: is not-permeable to a fluid FL; and it is capable of degrading at a temperature T; and the electromagnetically active layer is integrally attached to the support layer, is capable of converting electromagnetic energy carried by the incident electromagnetic radiation of wavelength W into thermal energy to produce the temperature T at which the sheathing layer degrades, thus producing a hole; and is thermostable at temperature T. The invention also provides processes for its preparation, uses as spectroscopy substrate, methods for identifying/quantifying one or more analytes and kits and devices comprising the substrate. Advantageously, the substrate of the invention allows the in situ detection or quantification of analyte(s) in real time and overcomes the "memory effect" reported with the spectrospic substrates known in the state of the art.

Description

Real Time Trace Detection

This application claims the benefit of European Patent Application EP20383131.8 filed December 21 , 2020.

Technical Field

The present invention relates to the field of spectroscopy, particularly of Raman spectroscopy. The present invention provides a layered substrate useful as Raman substrate, as well as methods for its preparation, methods for identifying or quantifying one or more analytes, and devices and kits containing said substrate.

Background Art

Raman spectroscopy is a powerful vibrational analysis technique that allows to give chemical and structural information on chemicals or biomolecules. Due to this property Raman is widely used in applications of biosensing, food control and detection of hazardous materials. Surface-enhanced Raman spectroscopy (SERS) uses the plasmonic property of noble metal particles, the SERS substrate, to enhance the Raman signal of molecules in the vicinity of such particles and attain detection limits that can go down to single molecule detection. Indeed, gold nanoparticles confine light to nanoscale volumes allowing for dramatic enhancement of the Raman scattering signal.

Real-time SERS detection would grant access to chemical information of a solution at a given time. Especially real-time SERS measurements in flow would allow for better water quality control. For instance, pesticide presence in tap water is a growing problem and real-time SERS analysis could allow for monitoring their presence. However, a major drawback arises for SERS measurements: when molecules adsorb on the surface of the plasmonic particles, their desorption becomes difficult. As a consequence, the Raman signal of such molecules is still visible even at later stages after incubation. Such an effect is known as the memory effect and impedes real time detection with the standard SERS strategy, where the analyte solution is continuously in contact with the plasmonic substrate. As a consequence, the main approach to monitoring changes in a solution consists in using a virgin substrate to detect the analytes at a given time. However, this strategy is not satisfactory enough as it does not allow for continuous SERS measurements in situ.

Different solutions have been proposed to render SERS substrates re-usable and therefore eligible for real-time sensing. The main strategy consists in cleaning the substrate from molecular adsorbates. Some cleaning techniques based on different chemicals or other treatments, such as UV-ozone, have shown good results for the re-use of plasmonic substrates for SERS measurements. Such techniques are interesting but they do not allow in-situ measurements, as the substrates need to be removed from the solution of interest to be cleaned. Beider and co-workers have shown real-time measurement in microfluidic channels by electrical regeneration of a silver wire (cf. Hohn et al., 2020). This method enables the monitoring of chemical species in real-time, however, it only gives a low SERS signal enhancement and it is hard to transfer to efficient plasmonic systems.

In spite of the efforts made, there is still the need of substrates which allow the successive in situ detection or quantification of analyte(s) in real time.

Summary of Invention

The present inventors have found that applying a sheathing layer with particular properties, over the whole plasmonic substrate surface, an efficient spatio-temporal control in the identification and/or quantification of the analyte(s) of interest can be achieved.

In particular, as it is shown below, the inventors applied a layer made of a thermolabile material, such as poly(lactic-co-glycolic acid) (PLGA), on a plasmonic substrate. This layer, which is not-permeable to the medium, was found to act as an efficient “shield” towards the fluid and analyte(s) present in the medium, avoiding any contact between the analyte(s) and the plasmonic substrate.

Surprisingly, it was found that when the laser of the Raman spectrometer was applied on this PLGA-coated substrate at a sufficient power density, the formation of a single microhole was obtained in the PLGA layer while the plasmonic substrate remained intact. The formation of a microhole in the PLGA layer renders the plasmonic substrate underneath accessible to the analyte to be detected just through that hole. The created hole enables the detection of the analytes in solution, while the remaining PLGA-coated surface of the plasmonic substrate remains in pristine conditions, therefore impeding contacting of the plasmonic layer with the analyte(s) thanks to the non-permeability of the sheathing layer.

Without being bound to the theory, the present inventors hypothesized that when the laser is applied on the substrate, the electromagnetic radiation of a suitable wavelength interacts with the electromagnetically active particles, the plasmonic particles, which convert the electromagnetic energy into thermal energy that gives rise to the local degradation of the sheathing layer. When the power density of the laser beam is suitably adjusted, a hole is created in the sheathing layer as a result of polymer degradation. Noteworthy, the temperature reached by the electromagnetic layer is sufficient to degrade the sheathing layer but does not affect the electromagnetically active layer. Thus, the area wherein the laser is applied on the SERS substrate of the invention gives rise to a hole in the sheathing layer while the electromagnetically active layer remains unaffected.

Consequently, the shielding layer allows to control the moment and particular area wherein the analyte and plasmonic substrate can contact one another: one hole, one measurement for a particular analyte. And, as the sheathing layer remains on the substrate, further holes and measurements can be taken. Advantageously, as it is illustrated below, at least 4 different analytes could be efficiently be identified on a 100x40 pm substrate (Fig. 4). These are promising results, as they would grant thousands of measurements on a substrate of 1 cm², which is a common size for a SERS substrate.

Until the present invention, SERS substrates came with a significant drawback: they did not allow for measuring changes over time in a solution, due to the “memory effect”. Indeed, once molecules adsorbed on a plasmonic surface, they could not be easily removed in general, and any subsequently added molecule could not be accurately sensed by the SERS substrate, as it did not interact with the plasmonic NPs in the same manner as with virgin substrates. This issue is a common problem in SERS and is very important when coming to real time detection or re-use of SERS substrates. Indeed, this drawback makes necessary the use of a new SERS substrate each time that a particular analyte has to be detected at a particular moment of time. Considering that commercial SERS substrates have a cost around 50 euros/unit on average, this means a significant economical drawback when measurements for long periods of time and for different analytes are required.

Advantageously, in the present invention, thanks to the coating with the not-permeable sheathing layer, this “memory effect” related to the use of known SERS substrates is also overcome, allowing for efficient real-time detection with a single SERS substrate. Fig. 1 provides a comparison between the results obtained with a plasmonic substrate of the state of the art (consisting of a plasmonic layer over a support layer, Fig. 1(a)) and with the plasmonic substrate of the invention (which in addition includes a not-permeable and thermolabile sheathing layer, Fig. 1(b)) in the detection of various analytes at different points in time. As it can be concluded, once the first analyte is added to the medium, the substrate of the prior art does not allow for the accurately detection of the subsequent added analytes. This is due to said “memory effect”: the incubation with the first analyte to perform SERS detection blocks the surface of the substrate, hindering the detection of the subsequent analytes. On the contrary, and following exactly the same protocol of addition of analytes, the substrate of the invention was able to detect each one of the analytes added to the medium. In addition to the above advantages, the substrate of the invention also shows a high capacity to detect the analyte in the hole. As it is shown below, SERS spectra from each hole showed characteristic peaks from the analyte present at the time of laser irradiation. This is also indicative of the fact that the plasmonic substrate and the sheathing layer are stable enough to appropriately play their role in spite of the successively created holes and SERS mappings.

As it is shown below, HeLa cells activity were monitored in a bioreactor comprising the substrate of the invention. To that end, the concentration of two metabolites, adenosine and hypoxanthine, were monitored for 24 hours. The results provided in Fig. 6(b) allow to conclude that the substrate of the invention is efficient and accurate in the continuous SERS in-situ measurement.

Altogether, the present invention means a great advancement in the accurate identification and monitoring of analyte(s): just applying a coating of a thermolabile, not-permeable material, on the SERS substrate to be used, the remarkable technical effects reported herein can be achieved.

Although the examples provided herein are based on the use of plasmonic substrates comprising a plasmonic layer adjacent to a support layer, the skilled person in the art knows that other substrates include, instead of a plasmonic layer, a dielectric or semiconductor layer. The same results are expected to be obtained because in all cases the role of the electromagnetically active layer, whether plasmonic, dielectric or semiconductor, is the same: converting electromagnetic energy carried by the incident electromagnetic radiation into thermal energy. The skilled person in the art also knows that energy conversion, from electromagnetic energy to thermal energy, is produced by the electromagnetic layer. This phenomenon takes place when the electromagnetic radiation reaches the electromagnetically active layer and it is independent on the radiation pathway. In other words, energy is successfully converted whether the radiation passes through the sheathing layer or through the support component.

Thus, in a first aspect the present invention provides a layered substrate comprising:

(a) an electromagnetically active layer,

(b) a support layer adjacent to the electromagnetically active layer, and

(c) a thermolabile sheathing layer adjacent to the electromagnetically active layer, wherein, at least one of the above-identified layers (b) and (c), adjacent to the electromagnetically active layer, is transparent to an incident electromagnetic radiation of wavelength W; the sheathing layer:

- is not permeable to a fluid FL; and

- is capable of degrading at a temperature T; and the electromagnetically active layer:

- is integrally attached to the support layer;

- is capable of converting electromagnetic energy carried by the incident electromagnetic radiation of the wavelength W, into thermal energy to produce the temperature T at which the sheathing layer degrades to generate a hole; and

- is thermostable at the temperature T at which the sheathing layer degrades.

In a second aspect, the present invention provides a process for the preparation of a layered substrate as defined in the first aspect of the invention, the process comprising the steps of:

(i) coating a support layer with an electromagnetically active layer, followed by

(ii) coating the resulting coated substrate of step (i) with a thermolabile sheathing layer, wherein the support layer, the electromagnetically active layer and the thermolabile sheathing layer are as defined above, and at least one of the electromagnetically active layer and the thermolabile sheathing layer is transparent to an incident electromagnetic radiation of wavelength W.

Although the experimental data provided below shows the usability of the layered substrate as a SERS substrate or biosensor, it will be understood that the substrate of the invention may be used in any suitable spectroscopy-based technique. For example, the substrate may be used as a biosensor substrate or a spectroelectrochemistry substrate. Such methods include: infrared spectroscopy, surface enhanced infrared absorption, surface plasmon resonance spectroscopy, photocatalytic and photovoltaic reactions, using all radiated energy; and photoelectron spectroscopy, including X-ray Photoelectron Spectroscopy (XPS), and Ultraviolet Photoelectron Spectroscopy (UPS).

Thus, in a third aspect the present invention provides the use of the substrate as defined in the first aspect of the invention as a sensor to detect, quantify and/or monitor one or more target analyte(s) contained in a fluid FL by a spectroscopy-based technique. In a fourth aspect the present invention provides the use of the substrate as defined in the first aspect of the invention as surface-enhanced Raman spectroscopy (SERS) substrate.

In a fifth aspect the present invention provides a method for identifying or quantifying one or more target analyte(s) contained in a fluid FL, the method comprising the steps of

(a) contacting a sample of the fluid FL with the sheathing layer of a layered substrate as defined in the first aspect of the invention; and

(b) obtaining a spectrum indicative of the amount or presence of the target analyte(s) in the sample, by:

(b.1) irradiating an area of the at least one of the transparent layers of the layered substrate with an incident electromagnetic radiation of wavelength W, particularly with a beam of electromagnetic radiation, to generate a hole in the sheathing layer; wherein the generation of the hole is performed by the electromagnetically active layer of the substrate, which converts the electromagnetic energy carried by the incident electromagnetic radiation of the wavelength W, passing through the transparent layer(s), into thermal energy, to produce the temperature T at which the sheathing layer locally degrades, thus producing the hole;

(b.2) irradiating the layered substrate resulting from step (b.1); and collecting the spectrum, particularly a SERS spectrum, indicative of the amount or presence of the target analyte(s) in the sample.lt has been widely reported that the functionalization of SERS substrates with particular reagents, such as antibodies, antigens, tags, aptamers, labelled and/or non-labelled nanoparticles, or detectable labels, among others, can significantly improve the level of sensitivity and selectivity, increase peak resolution by narrowing the width of the peaks, multiplex detection, and decrease photo bleaching as compared to fluorescence. On the other hand, the experimental data provided below show that measurements from different solutions containing different analytes, can be taken using a single substrate. Thus, the spatial control given by the hole creation in the PLGA layer could serve for regio-selective functionalization of plasmonic substrates, with molecules such as antibodies or aptamers in order to create a multiplexed assay, such as an immune-assay.

Thus, in a sixth aspect the present invention provides a method for functionalizing the layered substrate as defined in the first aspect of the invention with a reagent, the method comprising the steps of creating one or more holes in the sheathing layer of the substrate, and contacting the resulting substrate with a solution comprising the reagent. In a seventh aspect, the present invention provides a substrate obtainable by the method of the eighth aspect of the invention. The term "obtainable" and "obtained" have the same meaning and are used interchangeably. In any case, the expression "obtainable" encompasses the term “obtained”.

In an eighth aspect the present invention provides a device or kit for quantifying or detecting one or more analytes comprising a substrate as defined above.

Brief Description of Drawings

Fig. 1 - a) SERS spectra recorded on a control gold substrate (with no sheathing layer, - PLGA) after the sequential incubations with the following solutions: 1) 4-MBA 2) NAM and 3) a mixture of both analytes, 4-MBA+ NAM. b) SERS spectra recorded on the substrate of the invention, comprising a PLGA sheathing layer (+PLGA). Such spectra were acquired by measuring the intensity (I) in absorbance units (a.u.) of the signal at different Raman shift (k), after introducing the indicated solutions, with an excitation laser wavelength of 785 nm laser at normal incidence, 50x objective and a laser power of 0.03 mW/pm² power for 1s. The solid vertical line indicates the location of the 4-MBA peak while the location of the NAM signal is indicated by the dotted vertical line.

Fig. 2- a) Representative scanning electron microscopy (SEM) image (5 kV acceleration voltage) of gold superlattices substrate with a spin-coated (1500 rpm for 30s) layer of PLGA dissolved in acetone; the corresponding SERS spectra (785 nm excitation, 1s integration time, 0.03 mW/pm²) obtained after incubation with adenosine (100 pM) addedon top of the substrate is also provided (c). SERS spectra were recorded over different areas of the plasmonic substrate, heterogeneously displaying the intensity (I) in absorbance units (a.u.) of the characteristic adenosine vibration at 735 cm^-1 Raman shift (k); b) SEM image of the homogenous polymeric coating over the whole plasmonic superlattices substrate. This perfect coverage was obtained by spin-coating PLGA dissolved in ethyl acetate. In this case, SERS spectra (d) show no trace of the adenosine solution (100 pM) incubated on top of the substrate. Image and spectra were acquired as described in a).

Fig. 3 - SERS spectra of adenosine solution after each step of the hole creation process, the dotted lines parallel to the ordinates axis highlight the characteristic vibrational Raman shift (k) region of the adenosine molecule. All SERS measurements were acquired by recording the intensity (I) in absorbance units (a.u.) of the signal at the same point of the plasmonic substrate and using a 785 nm laser, a) Initial SERS spectra obtained by irradiation with 0.03 mW/pm² for 1 s. b) SERS spectra with 0.11 mW/pm² for 1 s laser irradiation, c) Final spectra obtained with 0.03 mW/pm² for 1 s, the same laser power as employed in the first step, d) SERS mapping at 735 cm^-1 of the previously irradiated area, this map is acquired with a laser power of 0.03 mW/pm²for 1s. e) SEM images of a hole created in the sheathing layer by laser irradiation, f) Gold nanoobjects located at the created hole (2) are exposed to analytes in solution. Regions (1) and (3) correspond to regions of the substrate with the PLGA coating, whereas region (2) correspond to the region wherein it is located the hole.

Fig. 4 - Multiplexing SERS experiment with high spatial resolution. A) Representative SERS spectra obtained over the holes created on the PLGA sheathing layer of a plasmonic substrate, as shown in B (1 , 4-MBA, dotted line indicates 1084 cm^-1; 2, Crystal Violet, dotted line indicates 1183 cm^-1; 3, Thiabendazole, dotted line indicates 1015 cm^-1; and 4, Nicotinamide dotted line indicates 1032 cm^-1). B) SERS map generated by integration of the characteristic modes of (1) 4-MBA at 1084 cm^-1, (2) Crystal Violet at 1183 cm^-1, (3) Thiabendazole at 1015 cm^-1 and (4) Nicotinamide at 1032 cm^-1.

Fig. 5 - SERS measurement acquired by using a 785 nm laser excitation at 0.11 mW/pm² irradiance for creation of a hole in the PLGA layer then of 0.03 mW/pm² for the Raman measurement. The plasmonic substrate was mounted on a microfluidic flow, and the fluid flow was provided by a syringe pump at 20000 pL/h. Subsequent introduction of water (W) or analyte solutions in the microfluidic channel was performed using a syringe pump. SERS intensity of the characteristic mode of thiabendazole at 1014 cm^-1 (T, black continuous line) and 4-MBA at 1080 cm^-1 (M, black dotted line), as a function of the introduction cycles (n) by the syringe pump, for a) a nanoobject superlattice without a PLGA sheathing layer, as a control and b) nanoobject superlattice coated with PLGA sheathing layer, (+PLGA). The black arrows on top of each graph represent the introduction of a different analyte solution (W, T or M) in the microfluidic channel at the indicated introduction cycle.

Fig. 6- a) SERS spectra of 100 pM Adenosine (Ado) and 100 pM Hypoxanthine (Hx) in aqueous solution. Dotted lines indicate the locations of the Ado peak at 735 cm^-1 and of the Hx peat at 725 cm^-1, b) SERS spectra recorded on control plasmonic superlattices without the PLGA sheathing layer (-PLGA) and plasmonic superlattices coated with the PLGA sheathing layer (+ PLGA) at different times (0 and 24 h) after Ado (200 pM) supplementation into the bioreactor, containing HeLa cells, c) SERS spectra of the extracellular supernatant extracted from the bioreactors and measured on fresh plasmonic superlattices. Such spectra were acquired with an excitation laser wavelength of 785 nm laser, 50x objective and a laser power of 0.03 mW/pm² power for 10 s. Fig. 7 - Schematic view of the methodology used to combine the silicone chamber (a) with the plasmonic superlattices on the top to perform the SERS measurements, passing the laser radiation through the support layer. For this purpose, gold nanoparticles-PLGA side of the plasmonic substrate must be oriented toward inner compartment (b). In c) the incorporation of a biological system, HeLa cells, inside the silicone chamber is represented, generating the corresponding bioreactor. Such a bioactive environment causes the conversion of Adenosine (Ado) to Hypoxanthine (Hx).

Detailed description of the invention

All terms as used in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly throughout the specification and claims unless an otherwise explicitly set out definition provides a broader definition.

For the purposes of the present invention, any ranges given include both the lower and the upper end-points of the range.

In a first aspect the present invention provides a layered substrate.

In the present invention, the term “adjacent” when referred to the layers of the substrate of the invention, means that they are in contact (as directly derivable from the process of the second aspect of the invention).

In the present invention, the term “integrally attached to” refers to the fact that discrete parts (i.e., the electromagnetically active and support layers) are physically joined together as a unit without each part losing its own separate identity when the substrate is in contact with a fluid F.

In the present invention, the ability of the electromagnetically active layer to be “capable of converting electromagnetic energy carried by the incident electromagnetic radiation of wavelength W into thermal energy” can be measured by detecting a change in the temperature of the electromagnetically active layer before and after applying the electromagnetic radiation on the substrate: if there is an increase in the temperature, this would mean that the layer is capable of converting the electromagnetic radiation into thermal energy. The temperature can be measured with an infrared thermal camara, such as an AX5 infrared thermal camera.

In the present invention the term “thermostable to temperature T”, when referred to the electromagnetically active layer, means that the electromagnetically active layer retains the surface topography and electromagnetic properties that it had before reaching temperature T, including the capability to convert electromagnetic radiation into thermal energy and the capability to enhance locally the electric field upon incidence of an electromagnetic radiation. The “temperature T” is the one acquired by the electromagnetically active layer in a particular irradiated area upon irradiation with an electromagnetic radiation to promote the degradation of the sheathing layer. If the temperature achieved in the irradiated area of the electromagnetically active layer is too high, however, it may cause damages, such as reshaping or degradation, and these can negatively affect the role of the electromagnetically active layer as sensor. Thus, it is mandatory that the electromagnetically active layer is structurally and functionally stable at temperature T. The surface topography can be confirmed by Transmission Electron Microscopy (TEM) or by Scanning Electron Microscopy (SEM), whereas the retaining of the functional role can be monitored by the characterization of the electromagnetic properties of the layer, for example the SERS signal detection properties.

In the examples provided below it is shown that the permeability of the sheathing layer can be modified due to heat, resulting in the interaction of the molecules with the plasmonic component. In particular, the heat generated by the plasmonic nanoparticles through light absorption and deexcitation of phonons was exploited to alter the permeability of the PLGA layer adjacent to the plasmonic layer. Consequently, by using a laser excitation with an elliptical spot size of 26x13 pm, it was found that there was an increase in local temperature to precisely degrade PLGA in the irradiated area. Such a plasmonic heating was validated with Infrared images as a local temperature increase from 30 to 38 °C on the plasmonic superlattice excited with a 785 nm laser at 0.11 mW/pm². No temperature increase was detected when the laser beam was focused on an area of the PLGA layer outside of the plasmonic superlattice, which validated that heating came from the plasmonic gold nanoparticles and not from the interaction of the laser with the PLGA layer.

In the present invention the term “permeability” to a fluid, when referred to the sheathing layer, refers to the ability of the layer to let the components of the fluid pass through. The components of the fluid can be solvent molecules alone or a combination of solvent molecules and other substances, including analyte molecules. An easy way to confirm whether the sheathing layer is permeable or not to the fluid involves contacting the layered substrate, having a sheathing layer of a thermolabile material coating over an electromagnetically active substrate, with a sample of the fluid, and performing a SERS analysis with a Raman spectrometer. In one embodiment the candidate sheathing layer is applied to the electromagnetically active substrate obtained as described in the examples below, sections 2.1 and 2.2., and the test of permeability comprises applying a drop of the fluid on the sheathing layer and then performing SERS readings with a laser radiation having a wavelength of 785 nm and an irradiance of 0.03 mW/pm² for a period of 1 second. Depending on the size of the substrate and the laser beam spot size, the test may have to be performed several times in random non-previously irradiated areas, for instance from 10 to 100 times, under criterium of the skilled person in the art. If a SERS signal is obtained, this will reveal that molecules from the fluid have diffused through the sheathing layer to the electromagnetically active layer, and therefore, that the sheathing layer is permeable. If no SERS signal is detected, this will be indicative that the sheathing layer is not-permeable to the fluid. The particular combination of irradiance and irradiation time pointed out above makes the incident electromagnetic radiation innocuous to the sheathing layer, that is, it does not have any effect on the permeability properties of the sheathing layer that translates into a detectable SERS signal.

In the present invention, the term “thermolabile”, when referred to the sheathing layer, means that the layer is “capable of being degraded at a temperature T”. The particular temperature T at which the thermolabile sheathing layer degrades is produced by the electromagnetically active layer upon irradiation of the substrate with an electromagnetic radiation. It is known to the person skilled in the art that different combinations of wavelength, irradiance and irradiation time can produce the same temperature when applied to a given substrate. On the other hand, such combinations will need adjustment to reach the same temperatures with different substrates. Additionally, the same amount of electromagnetic energy delivered to the substrate in varying periods of time will result in different temperatures since thermal dissipation of the substrate will counter effect the local conversion of electromagnetic energy into thermal energy. The degradation of the sheathing layer at a temperature equal or above said temperature T gives rise to a local change in the sheathing layer resulting in the destruction of the layer and production of a hole in the area of the sheathing layer in contact with the irradiated area of the electromagnetically active layer, allowing for the diffusion of fluid molecules toward the electromagnetically active layer (wherein detection occurs). In one embodiment, the degradation is irreversible. Thus, the ability of degrading the sheathing layer can be indirectly measured by monitoring changes in the permeability, following the protocol provided above.

In the present invention the term “fluid” refers to a liquid or a gas, in which the analyte molecules are dispersed/dissolved and transported. In one embodiment the fluid is a liquid. Alternatively, in another embodiment the fluid is a gas. In another embodiment, the fluid is a biological fluid, such as plasma, serum, urine, or blood, or a fluid from industrial origin (such as contaminated water).

In the present invention, the term “transparency” is the physical property of materials to let light pass through without or with low absorption. Such a property is measured by LIV-VIS spectroscopy, determining the absorbance: A= log (lo/l) wherein I is the intensity of light after it passed through the sample and Io is the intensity of light before passing through the material. In the context of the present invention, a material is considered to be transparent to a specific wavelength W if its absorbance (A) is within the range comprised from 0 to 3, particularly from 0 to 2 or from 0 to 1. In one embodiment, the sheathing layer is transparent to the electromagnetic radiation of wavelength W. In this embodiment, the irradiation is performed through this layer. In another embodiment, the support layer is transparent to the electromagnetic radiation. In this alternative embodiment the radiation can be performed through the support layer.

In one embodiment of the first aspect of the invention, the electromagnetically active layer converts the electromagnetic energy into thermal energy to a temperature T upon irradiation with an electromagnetic radiation of wavelength W.

There are well-known techniques and devices in the state of the art to confirm whether the electromagnetic layer is capable of producing a temperature “T”. In one embodiment, the capability of producing a temperature T is determined using an AX5 infrared thermal camera.

In another embodiment of the first aspect of the invention, the electromagnetically active layer produces a temperature T upon irradiation with an electromagnetic radiation of wavelength W, delivered at an irradiance P during an appropriate period of time. The “appropriate period of time” when referred to the irradiance, is understood as the irradiation time needed to obtain a fluence F.

In the present invention, the term “irradiance”, also known as “Flux density”, refers to the radiant flux, radiant energy, radiant power or simply power (abbreviated as “P”) received by a surface per unit area. In this invention, irradiance is normally expressed in mW/ pm². In one embodiment, the irradiance is within the range from 10'⁶to 10⁸ mW/pm². In one embodiment, the irradiance is equal or below 10 mW/pm², particularly comprised from 10'⁶ to 10 mW/ pm² In another embodiment, the irradiance is comprised from 0.01 to 10 mW/pm², particularly from 0.01 to 1 mW/pm² and the irradiation time is in the range from 1 ms to 60s, particularly from 1 to 10 s. In an alternative embodiment, the irradiation time can be a short pulse of femtoseconds. In this alternative embodiment the irradiance can be within the range from 1 ■ 10³ to 1 ■ 10⁸ mW/pm².

In the present invention, the term “fluence” (abbreviated as “F”, expressed in mJ/pm²) is understood as the energy (in J) delivered by a radiation source to the sample during a given period of time, divided by the irradiated area. The energy is calculated by integrating the power of the laser, measured using a power meter (Ophir TE head), over the irradiation time period (pulse). In this invention the irradiated area is calculated by measuring the spot size of the laser beam or by measuring the size of the hole generated in the sheathing layer by scanning electron microscopy (SEM). In one embodiment, the fluence F corresponds to the value that makes the electromagnetic active layer to reach temperature T.

It should be noted that the same fluence can be achieved with different irradiance values by varying the irradiation time. Therefore, the irradiation of a given surface capable of converting electromagnetic energy into thermal energy, with the same fluence, can lead to different temperatures depending on the heat dissipation properties of the surface and of the environment surrounding the surface. The person skilled in the art will realise that by adapting the irradiance and irradiation time it is possible to achieve comparable amounts of thermal energy delivered by the irradiated surface per unit area and that this thermal energy will produce the target temperature or a temperature-dependent effect when the irradiation is provided in a sufficiently short period of time as to counter effect the thermal dissipation of the irradiated substrate.

In the context of the present invention, the “electromagnetically active layer” means a layer capable of enhancing the electromagnetic field of an incident electromagnetic radiation. This phenomenon makes the substrate of the invention suitable as spectroscopic sensor, being capable of enhancing the spectroscopic signal of a target analyte contained in a fluid FL. The enhancement in the spectroscopic signal, which provides an indirect information about the capability of enhancing the electromagnetic field, can be determined by measuring the spectroscopic signal, particularly the SERS signal, of a target analyte contained in a fluid FL with and without the incidence of an electromagnetic radiation field having a wavelength W, for example a laser radiation having a wavelength of 785 nm and an irradiance equal or below 10 mW/pm²for a period from 1 ms to 60 seconds or, alternatively, applying a femtopulse of a radiation with an irradiance from 10³ to 10⁸. If the signal provided by the tested analyte is increased in a factor comprised from 10 to 10¹², this will be indicative that the tested electromagnetically layer is, effectively, an electromagnetically active layer capable of appropriately enhancing the electromagnetic field and the spectroscopic signal of the target analyte. In another embodiment of the first aspect of the invention, the sheathing layer comprises one or more materials selected from the group consisting of polymers, block-copolymers, proteins and other biomolecules, metal organic frameworks, covalent organic frameworks, organic molecular films and the like. In another embodiment of the first aspect of the invention, the sheathing layer comprises thermolabile polymeric chains. In another embodiment of the first aspect of the invention, the sheathing layer consists of thermolabile polymeric chains. In another embodiment, the thermolabile polymeric chains are selected from: polyacrylates (such as polyethyl, polybuthyl), polymetacrylates (such as poly methyl metacrylate), polyvinyl acetates, polyvinyl acetate copolymers (such as poly ethylene vinyl acetate), poly(lactic acid) polymers, and poly(lactic-co-glycolic acid) copolymers.

In another embodiment, the thermolabile polymeric chains are selected from the group consisting of: poly(lactic acid) polymers, poly(lactic-co-glycolic acid) copolymers, particularly poly(lactic-co-glycolic acid) copolymers, and combinations thereof. Advantageously, the degradation of PLGA (o related polymers) in aqueous environments upon irradiation generates species (lactic and glycolic acid) that are biocompatible.

In another embodiment, the sheathing layer does not comprise plasmonic particles.

In another embodiment, the thermolabile polymeric chains are PLGA copolymers. Illustrative non-limitative examples of PLGA copolymers are those comprising 75:25, 85:15 or 95:5 (molar ratio lactide:glycolide). In another embodiment, the sheathing layer consists of PLGA polymeric chains. In another embodiment, the sheathing layer consists of PLGA polymeric chains 75:25, 85:15 or 95:5.

In another embodiment, the sheathing layer has a thickness equal to or larger than the thickness of the electromagnetically active layer, particularly it has a thickness up to 10 pm, particularly from 0.5 to 10 pm, particularly from 0.8 to 2 pm.

The thickness of any of the layers forming part of the substrate of the invention can be measured using well-known means, such as transmission electron microscopy (TEM) or scanning electron microscopy (SEM).

In one embodiment, the substrate is wholly coated with a sheathing layer consisting of a PLGA layer having a thickness from 0.5 to 10 pm, particularly from 0.8 to 2 pm.

In another embodiment, the electromagnetically active layer comprises one or more materials selected from the group consisting of metallic materials, semiconductor and dielectric materials.

As it has been explained above, the electromagnetically active layer is responsible for converting incident electromagnetic radiation at a particular wavelength W into heat. The conversion of light to heat can be monitored using a thermal camera which can record the temperature prior and during radiation incidence. The efficiency of light-to-heat conversion strongly depends on the optical absorption properties of the electromagnetically active layer. For an optimal light to heat conversion, the electromagnetically active material should be chosen to absorb a maximum amount of light at the wavelength W of the excitation laser, to promote the heating of the layer to a temperature T, said temperature being responsible for locally breaking the sheathing layer, thus producing a hole, without negatively affecting the functionality of the electromagnetically active layer.

The skilled person in the art can easily select the most appropriate material(s) to be used in the electromagnetically active layer, on the basis of the thermolabile material forming the sheathing layer (which degrades at a particular temperature T). Depending on the temperature of degradation of the sheathing layer, the skilled person can select the appropriate electromagnetically active material. For example, the skilled person in the art knows that if it is required that the electromagnetically active layer promotes the generation of high temperatures (such as 150°C) for degradation of polymers such as polypropylene carbonate or polymethyl methacrylate, aggregated gold nanospheres should be selected (Borah R. et al., 2019). Alternatively high temperatures can be attained using iron oxide nanoparticles (Zograf G. P. et al., 2020). If moderate temperatures are required (such as 60°C) for degradations of polymers like polyethylene terephthalate, polymethylpentene, then single gold nanoparticles can be selected, especially gold nanorods or nanostars.

In another embodiment, the electromagnetically active layer is in the form of a particulate layer or of a continuous layer (i.e. , a continuous film), particularly in the form of a particulate layer. The form as particulate or continuous layer can be easily determined by TEM or SEM. Thus, it can be seen that when the electromagnetically active layer is in the form of a particulate layer, the obtained images show nanoobjects, made of the electromagnetically active material, distributed along the support layer (see for instance FIG. 2).

In the present invention, the term “nanoobject” refers to a primary particle (nonagglomerated single particle) with one, two or three external dimensions in the nanoscale, as recognized by the International Organization for Standardization in the document with the reference number ISO/TS 27687:2008(E). Illustrative non-limitative examples of nanoobjects are: nanoparticles, which are nanoobjects with all three external dimensions in the nanoscale (if the lengths of the longest to the shortest axes of the nanoobject differ significantly, typically by more than three times, the terms nanofibre or nanoplate are intended to be used instead of the term nanoparticle); nanosheets (or nanoplates or nanolayers), which are nanoobjects with one external dimension in the nanoscale and the two other external dimensions significantly larger, wherein the smallest external dimension is the thickness of the nanosheets, the two significantly larger dimensions are considered to differ from the nanoscale dimension by more than three times, and the larger external dimensions are not necessarily in the nanoscale; nanofibres, which are nanoobjects with two similar external dimensions in the nanoscale and the third dimension significantly larger, wherein the nanofibres can be flexible or rigid and the two similar external dimensions are considered to differ in size by less than three times and the significantly larger external dimension is considered to differ from the other two by more than three times, and the largest external dimension is not necessarily in the nanoscale; nanotubes, which are hollow nanofibres; nanorods, which are solid nanofibres; nanowires, which are electrically conducting or semiconducting nanofibres; and quantum dots, which are crystalline nanoparticles exhibiting size-dependent properties due to quantum confinement effects on the electronic states.

In one embodiment, the plasmonic nanoobjects are selected from nanospheres, nanosheets, nanorods, nanoprisms, nanostars, nanowires, nanofibers, and nanotubes.

The term “object size” when referred to the nanoobjects refers to a characteristic physical dimension of the primary particle. For example, in the case of a spherical nanoobject, the “object size” corresponds to the diameter of the nanoobject. In the case of a rod-shaped nanoobject with a circular cross-section, as it is the case of a nanofibre (either as such or in the form of a nanowire or nanotube), the “object size” of the nanoobject corresponds to the diameter of the cross-section of the nanoobject. In the case of a box-shaped nanoobject, such as a nanosheet, a nanocube, a nanobox, or a nanocage, the size of the nanoobject corresponds to its thickness. When referring to a set of nanoobjects being of a particular size, it is contemplated that the set of nanoobjects can have a distribution of sizes around the specified size.

The size of the nanoobjects of the invention can be determined using well-known techniques in the state of the art such as Transmission Electron Microscopy (TEM). Images are chosen to be as representative of the bulk sample as possible. The measured dimension is chosen depending on the morphology of the nanoobject as described above. In one embodiment, the nanoobjects have a size from 1 to 1000 nm. In some embodiments, the size is in the range of about 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, or 10-100 nm, all subunits included. In one embodiment, the nanoobjects are nanospheres with a size comprised from 5 to 100 nm, particularly from 10 to 50 nm. The wavelength of light in resonance with a plasmon mode in the nanoparticles will vary with the size and shape of the nanoparticles.

The density of the nanoparticles on the substrate layer may vary depending on factors such as the substances to be detected and the production process for the nanoparticles. Non-limiting examples of nanoobject density include about 10-2000, particularly from 10- 800 nanoobjects/pm², all subunits and sub-ranges included. Other examples include about 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450 and 500 particles/pm². In the examples provided below, the density was comprised from 100 to 200 nanoobjects/ pm². The nanoobjects can include protrusions of various shapes on the surface to further modify the plasmonic resonance properties of the nanoparticles. For example, the protrusion may be in the shape of columns, cones, tips, ridges, or combination thereof. In another embodiment, the nanoobjects are rough. Alternatively, the nanoobjects can be substantially or completely smooth (i.e. , with no protrusions).

In one embodiment, the electromagnetically active layer is in the range from 1 nm to 300 nm, all subunits included. Exemplary embodiments of the thickness of the coating include about 1 , 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 250 and 300 nm.

In another embodiment, the electromagnetically active layer is a plasmonic layer. In another embodiment the plasmonic layer comprises at least one of the materials included in the group consisting of noble metals, transition metals, metal oxides such as noble and transition metal oxides, alkaline earth metal oxides, alloys and combinations thereof. In another embodiment, the plasmonic layer comprises a metal selected from the group consisting of silver (Ag), osmium (Os), palladium (Pd), platinum (Pt), gold (Au), rhodium (Rh), ruthenium (Ru), rhenium (Re), iridium (Ir) or any combinations or alloys thereof; a transition metals include iron (Fe), copper (Cu), nickel (Ni), zinc (Zn), cobalt (Co), manganese (Mn), chromium (Cr), molybdenum (Mo), tungsten (W), aluminium (Al), tin (Sn), alloys and combinations thereof. In another embodiment, the plasmonic layer comprises gold. In another embodiment, the plasmonic layer is a particulate plasmonic layer comprising plasmonic nanoobjects, which can be the same or different. In another embodiment, the plasmonic nanoobjects are the same or different and comprise a metal selected from the group consisting of Ag, Au, Cu, Al, Mg, Ni, Pd, and Pt, alloys and combinations thereof. In another embodiment, the plasmonic layer consists of a particulate gold layer. In another embodiment, the plasmonic layer consists of gold nanospheres.

As it is shown below, gold nanoparticles convert light to heat by deexcitation of absorbed light through phonons. On this account, the selected plasmonic material with aggregated gold nanoobjects can give rise to more intense temperatures than single gold nanoparticles under laser excitation.

Thus, in another embodiment of the first aspect of the invention, the metallic nanoobjects are aggregated metallic nanoobjects. The present invention provides in the examples below an illustrative example of how gold aggregates can be obtained, based on well-known protocol based on successive growing steps using HAuCk, L-ascorbic acid and cetyl-tri- methyl-ammonium chloride. The skilled person in the art knows other ways to obtain aggregates with other plasmonic, dielectric and semiconductor materials. It is routine the selection of the more appropriate protocol to arrive to such aggregates. Given the fact that a gold nanoobject superlattice modulates the maximum absorption wavelength through periodicity of the lattice, they are good candidates as light to heat converters (Matricardi C. et al., 2018).

Thus, in another embodiment, the plasmonic layer comprises particulate metal nanoobjects homogenously distributed along the support layer. In another embodiment, the plasmonic layer consists of metal nanoobjects homogenously distributed along the support layer. In another embodiment, the plasmonic layer comprises gold nanoobjects homogenously distributed along the support layer. In another embodiment, the plasmonic layer consists of gold nanoobjects homogenously distributed along the support layer. In another embodiment, the plasmonic layer comprises gold nanospheres homogenously distributed along the support layer. In another embodiment, the plasmonic layer consists of gold nanospheres homogenously distributed along the support layer.

The term “homogenously distributed”, when referred to the nanoobjects forming part of the particulate electromagnetically active layer, means that these nanoobjects are periodically distributed in the X-Y plane of the support layer. This periodical distribution can be confirmed by TEM or SEM imaging. As an illustrative example of nanoobjects homogenously distributed along the support layer, reference is made to FIG. 2. This figure, which corresponds to a TEM/SEM of the gold coating over the poly(dimethylsiloxane)PDMS support layer, shows periodic dots, corresponding to the nanoobjects. The periodicity of the lattice improves the profile of the electromagnetically active layer as light to heat converters.

Alternatively, anisotropic plasmonic nanoobjects such as anisotropic gold nanoparticles, thanks to high heat localization at their tips, could offer more efficient heating than nanospheres in other experimental setups.

In one embodiment, the nanoobjects are surface functionalized, thus improving their adhesion to the support layer. The skilled person in the art knows that the nature of the functionalization depends on the material of the nanoobject: (i) noble metals like Au and Ag are normally functionalized with thiols or, to a lesser extent, amines and cyanides; (ii) oxides can be easily coated via oxygen bonding with acidic and hydroxyl groups; (iii) binary compounds, particularly those including elements from Groups 12 to 16 as components of fluorescent semiconductor (SC) NPs (e.g., quantum dots), display high affinity towards thiols and hydroxyl groups, but also amino groups are often used. There are also many suitable polymeric ligands, such as poly(ethylene glycol) (PEG). In one embodiment the nanoobjects are surface functionalized with PEG. In another embodiment, the nanoobjects comprise a plasmonic material and are surface functionalized, particularly with PEG. In another embodiment, the nanoobjects are plasmonic nanospheres surface functionalized, particularly with PEG. In another embodiment, the nanoobjects are gold nanospheres surface functionalized, particularly with PEG.

In one embodiment of the first aspect of the invention, the plasmonic nanoobjects have one, two, three, four or all the following features:

(i) are selected from nanospheres, nanosheets, nanowires, nanofibers, and nanotubes;

(ii) are aggregated metal nanoobjects;

(iii) are the same or different and comprise one or more metals selected from the group consisting of silver (Ag), osmium (Os), palladium (Pd), platinum (Pt), gold (Au), rhodium (Rh), ruthenium (Ru), rhenium (Re), iridium (Ir), iron (Fe), copper (Cu), nickel (Ni), zinc (Zn), cobalt (Co), manganese (Mn), chromium (Cr), molybdenum (Mo), tungsten (W), aluminium (Al), tin (Sn), alloys, and any combination thereof;

(iv) are surface functionalized, such as PEG-surface functionalized; and

(v) are homogenously distributed on support surface.

Alternatively to the plasmonic materials, the electromagnetically active layer can comprise a semiconductor and/or dielectric material. In one embodiment, the dielectric material is selected from the group consisting of silicon, germanium, carbon, and/or lll-V semiconductor materials.

In another embodiment, the support layer is rigid or flexible. In one embodiment, the support layer is flexible.

In the present invention, a substrate is defined as being “flexible” by determining the “bending cycle”. The “bending cycle” means the bending of flexible substrate convexly or concavely with a given radius, r, with respect to the base platform, and optionally, bending the substrate back to being flat. The bending cycle can be repeated. The curvature of the flexible substrate can be slight (e.g., two ends of the substrate just off from the horizontal plane of the substrate) to the ends touching upon the bending. The term “concave bending” means bending of the flexible substrate using a sequence of decreasing radii with respect to the base platform. The term “convex bending” refers to the bending of the flexible substrate using a sequence of increasing radii with respect to the base platform.

Suitable flexible substrates include substantially clear and transmissive polymer films, reflective films, transflective films, polarizing films, multilayer optical films, metallic films, metallic sheets, metallic foils, and the like. Flexible substrates can also be coated or patterned with electrode materials or transistors, for example transistor arrays formed directly on the flexible substrate or transferred to the flexible substrate after being formed on a temporary carrier substrate. Suitable polymer substrates include polyester base (e.g., polyethylene terephthalate, polyethylene naphthalate), polycarbonate resins, polyolefin resins, polyvinyl resins (e.g., polyvinyl chloride, polyvinylidene chloride, polyvinyl acetals, etc.), cellulose ester bases (e.g., cellulose triacetate, cellulose acetate), and other conventional polymeric films used as supports. For making organic electroluminescent devices on plastic substrates, it is often desirable to include a barrier film or coating on one or both surfaces of the plastic substrate to protect the organic light emitting devices and their electrodes from exposure to undesired levels of water, oxygen, and the like.

In another embodiment of the first aspect of the invention, the support is transparent to the radiation having a wavelength “W”. Advantageously, the irradiation can take place from the side of the device opposite to the sheathing layer. This is relevant because it allows the placement of the sensor in the inner side of the wall or a large vessel while irradiating from outside the vessel.

Any of the currently available materials commonly suitable as support in the manufacture of SERS substrates can be used. In one embodiment, the support layer comprises a material selected from the group consisting of silicon, silicon-based polymer, glass, silicon nitride, quartz, ceramics, sapphire, a plastic, and combinations thereof. In another embodiment, the polymer is selected from the group consisting of a polystyrene, polycarbonate, polyethylene, silicon-based polymer and combinations thereof. In another embodiment, the support layer comprises a silicon-based polymer. In another embodiment, the support layer consists of a silicon-based polymer. In another embodiment, the support layer comprises polydimethylsiloxane (PDMS). In another embodiment, the support layer consists of PDMS.

In one embodiment, the electromagnetically active layer is directly and integrally attached to the support layer. In an alternative embodiment, the electromagnetically active layer is indirectly and integrally attached to the support layer by means of an interposed layer, particularly an interposed adhesive or metallic layer.

The interposed layer may influence the adhesion of the electromagnetically active layer to the support layer. Typically, the interposed layer has high thermal resistance (i.e. , it does not degrade at the temperature T reached in the electromagnetically active layer).

Suitable interposed layers include, for example, polymer films, metal layers (e.g., vapor deposited metal layers), inorganic layers (e.g., sol-gel deposited layers and vapor deposited layers of inorganic oxides (e.g., silica, titania, and other metal oxides), and organic/inorganic composite layers. Suitable organic materials include both thermoset and thermoplastic materials. Suitable thermoset materials include resins that may be crosslinked by heat, radiation, or chemical treatment including, but not limited to, crosslinked or cross-linkable polyacrylates, polymethacrylates, polyesters, epoxies, and polyurethanes. Suitable thermoplastic materials include, for example, polyacrylates, polymethacrylates, polystyrenes, polyurethanes, polysulfones, polyesters, and polyimides. These thermoplastic organic materials may be applied via conventional coating techniques (for example, solvent coating, spray coating, or extrusion coating). The interposed layer may be either transmissive, absorbing, reflective, or some combination thereof, at the electromagnetic radiation wavelength.

Inorganic materials suitable as interposed materials include, for example, metals, metal oxides, metal sulfides, and inorganic carbon coatings, including those materials that are highly transmissive or reflective at the electromagnetic radiation wavelength. These materials may be applied to the light-to-heat-conversion layer via conventional techniques (e.g., vacuum sputtering, vacuum evaporation, or plasma jet deposition).

The thickness of the interposed layer may depend on factors such as, for example, the material of the interlayer, the material and properties of the electromagnetically active layer and the wavelength of the incident radiation, among others. For polymer interposed layers, the thickness typically is in the range of 0.05 pm to 10 pm. For inorganic interposed layers (e.g., metal or metal compound interposed layers), the thickness is in the range of 0.005 pm to 10 pm. Multiple interposed layers can also be used; for example, an organic-based interposed layer can be covered by an inorganic-based interposed layer to provide additional protection to the transfer of heat.

In another embodiment, the substrate further comprises an adhesive layer on the side of the support layer opposite to the side adjacent to the electromagnetically active layer. In this way, the substrate can be adhered to the wall of the device wherein the detection has to be performed.

Useful adhesive materials to be used are well-known and commonly used by the skilled person in the art. Illustrative non-limitative examples are polyethylenimine, polyvinyl acetate, phenol formaldehyde, and ethylene vinyl acetate, among others.

In another embodiment the substrate of the invention has a solid-planar conformation.

In the present invention, the term “solid” conformation means that the substrate has no gaps between the different layers. In a second aspect, the present invention provides a process for the preparation of a layered substrate as defined in the first aspect and any one of the embodiments provided above, the process comprising the steps of:

(a) coating an electromagnetically active layer with a thermolabile and not permeable sheathing layer; or, alternatively,

(ii) coating the resulting coated substrate of step (i) with a thermolabile sheathing layer, wherein the support layer, the thermolabile sheathing layer, the electromagnetically active layer and the support layer, are as defined in any of the embodiments provided above.

All of the embodiments provided under the first aspect of the invention, regarding the SERS substrate, are also embodiments of the method of the second aspect of the invention.

The fabrication of the electromagnetically active substrate generally follows different techniques that can be separated in two categories: top-down or bottom-up. In top down approaches the material is etched to create a nanostructure while bottom-up approaches involve the assembly of single atoms, molecules or clusters to yield a nanostructured material.

One of the most commonly used bottom-up approaches is the synthesis of nanoobjects and their subsequent assembly into support layers. The synthesis of nanoobjects of metals (cf. Hanke C. et al., 2017; Lu X. et al., 2009; Quintanilla M. et al., 2019), dielectrics (cf. Quan B., 2019), or semiconductors (cf. Norris D. et al., 1996) is well known and routine, and nanoobjects of almost all materials can be synthesized in different sizes and shapes. Such nanoobjects can be deposited on substrates by means of different techniques such as dropcasting (cf. Park J. et al., 2006), but more sophisticated techniques such as capillary self-assembly are required to get nanostructures with better organization. Capillary self-assembly enables the assembly of nanoobjects regardless of their material (cf. Flauraud V. et al., 2007) and the nanoobject size. Other illustrative non- limitative examples of suitable techniques are templated self-assembly, droplet, electrostatic self-assembly, vapour deposition, sputtering, photo and electron lithography, laser writing and 3D-printing, and combinations thereof.

Using top-down approaches, either a film or nannoobjects of the desired material are deposited on a substrate layer, and then etched away to retrieve the desired structure or or the material itself is deposited on a template that can be selectively removed to get the nanomaterial. In either case, the material film is generally obtained by sputtering, thermal evaporation, chemical vapour deposition or epitaxial growth (cf. Jilani A. et al., 2017).

The binding of a thin film of the electromagnetically active layer on the support layer, largely depends on the chemical properties of the material and its interaction with the substrate. Commonly used substrates for the fabrication of electromagnetically active substrate are glass or silicon wafers, which have similar surface properties. The adhesion of metals to glass substrates can be considerably improved by adding a layer of chromium or titanium. Chromium binds strongly to glass and gold binds strongly to chromium (cf. Colas F. et al., 2015). Thin layers of chromium or titanium can be deposited using sputtering or thermal evaporation. For other metals, the use of covalent bonding between the metal and thiol can be used for immobilizing metals on glass functionalized with thiols. For other materials, especially polar materials, creation of hydroxyl groups on the glass surface by plasma activation greatly improves the binding of the film of particles to the glass (cf. Pasternak L. et al., 2018; Xu J. et al., 2018). There also exist the possibility to coat the substrate layer with an adhesive coating. This can be performed by incubating the substrate layer in a solution of the adhesive. For instance, glass or PDMS can be coated with polyethyleneimine by incubating the PDMS with a polyethyleneimine (PEI) solution for 2 hours. PEI is a well-known adhesive and enables nanoobject to stick on the PDMS substrate (cf. Steiner A. et al., 2017).

For the case of nanoobjects, similar techniques to the ones cited before can be used, but there also exists the possibility of functionalizing, either the surface of the support or of the nanoobjects with a polymer that can interact, respectively, with the nanoobjects or the support layer. Either the polymer has a functional group that interacts with the bare substrate layer or the substrate is functionalised with a polymer that interacts with the nanoobjects or with the polymer functionalizing the nanoobjects. Another possibility is to apply a plasma process once the functionalized nanoobjects are deposited on the substrate layer. In the case of polyethyleneglycol (PEG) coated nanoobjects, the plasma process induces a better adhesion of the nanoobjects onto the glass layer (cf. Plou J. et al., 2020). For metallic nanoobjects, it is possible to functionalize the glass layer with a molecule comporting both a silanol and a thiol function, so that the silanol creates a covalent bond with the glass substrate and the thiol group is available for the creation of a covalent bond with metallic nanoobjects (cf. Kinnear C. et al., 2018). Other strategies include the functionalization of the substrate with a charged polymer to attract nanoobjects through electrostatic interactions (cf. Su Q. et al., 2011).

In one embodiment of the process of the second aspect of the invention, when the electromagnetically active layer comprises metallic nanoobjects, optionally surface functionalized (for example with PEG), then step (i) is performed by casting a metallic nanoobject dispersion on one side of the support layer. In another embodiment of the process of the second aspect of the invention, the casting performed in step (i) comprises applying a droplet of the metallic nanoobject dispersion on the surface of the substrate and subsequently application of a stamp on the droplet.

The deposition of the thermolabile polymer can be done using well-known and routine techniques. Illustrative non-limitative examples are: spin coating, dip coating, layer by layer assembly, sol-gel deposition, Langmuir-Blodgett, and vapour deposition. In one embodiment, step (i) or (ii) are performed by dip coating. Advantageously dip coating can work even for non-planar objects, and has been reported as efficient in providing uniform polymeric coating of metallic screws (Kim Y. K. et al., 2018). Spin coating is a routine technique for the fabrication of uniform thin films of photolithography, electron lithography resists and hot embossing. There are commercially available devices to perform this coating and only the speed needs to be adjusted as a parameter. The skilled person knows that to perform the dip coating it is required to pour a solution comprising the thermolabile polymer in a recipient, the substrate is then dipped in this solution at speed controlled by a device.

In an embodiment of the second aspect of the invention, the process further comprises a step (1) comprising irradiating an area of the substrate resulting from step (a) or (ii) with an incident electromagnetic radiation of wavelength W, and, optionally, a step (2) wherein the resulting substrate is functionalized with a reagent. Optionally, step (1) can be successively repeated to create several holes in the sheathing layer, previously to the functionalization with the reagent.

In a fourth aspect the present invention provides the use of the substrate of the invention as a SERS substrate.

Surface-enhanced Raman scattering (SERS) is a powerful vibrational analysis technique that allows to give chemical and structural information on chemicals or biomarkers. Raman scattering relies on the inelastic scattering of light by a molecule, especially each chemical bond is characterized by the frequency shift between incoming light and scattered Raman light generally expressed in Raman shift wavenumber, k, and cm^-1. The Raman signal can be enhanced using an electromagnetically active substrate, thus referring to surface-enhanced Raman spectroscopy or surface-enhanced Raman scattering (SERS). The enhancement of the Raman signal relies on two phenomena: (i) the enhancement of the electric field-in the vicinity of the electromagnetically active layer and (ii) charge transfer from the electromagnetically active layer to the adsorbed molecule. Generally, the largest part of the enhancement comes from the increase of electric-field in the near proximity of the electromagnetically active layer. Such an enhancement depends on the material and nanostructure used, it is quantified by the following equation:

with Eioc the local electric field , Esca the scattered electric field , sca the wavelength of Raman scattering, and A_exc the wavelength of excitation.

In a fifth aspect, the present invention provides a method for identifying or quantifying one or more target analyte(s) contained in a fluid FL, the method comprising the steps of (a) contacting a sample of the fluid FL with the sheathing layer of a substrate as defined in the first aspect; and (b) obtaining a spectrum indicative of the amount or presence of the target analyte(s) in the sample.

All of the embodiments provided under the first aspect of the invention, regarding the substrate, are also embodiments of the method of the fifth aspect of the invention.

In one embodiment of the fifth aspect of the invention, step (b.2) is performed with an incident radiation the same or different from the one used in step (b.1 ), depending on the particular analyte(s) to be detected or quantified and on the spectroscopic technique used

The spectrum can be collected using any of the suitable spectroscopy techniques, such as infrared spectroscopy, surface enhanced infrared absorption, surface plasmon resonance spectroscopy, photocatalytic and photovoltaic reactions, photoelectron spectroscopy, including X-ray Photoelectron Spectroscopy (XPS), and Ultraviolet Photoelectron Spectroscopy (UPS), among others. In one embodiment of the fifth aspect of the invention, step (b) comprises the steps of: (b.1) irradiating an area of the substrate with an incident electromagnetic radiation of wavelength W; and (b.2) collecting the SERS spectrum.

In one embodiment of the fifth aspect of the invention, the generation of the hole in the sheathing layer, and the reading of the analyte in the electromagnetically active layer are performed with the same radiation source, having the same fluence F.

In an alternative embodiment, step (b.1) is performed with a radiation having a fluence F higher than the one of the radiation used in the second step (b. 2). Thus, in the step wherein the hole is generated the substrate of the invention is irradiated with a higher energy radiation, to give rise to the creation of the hole in the sheathing layer and then, the reading is performed using a lower energy (i.e. , a lower fluence), such that the analyte is detected without creating unnecessary holes on the sheathing layer. In this way the re- usability of the substrate can be optimized. In the examples provided below, wherein the substrate comprises a gold layer, step (b.1) is performed with a fluence of 0.11 mJ/pm², whereas step (b.2) is performed at a fluence 0.03 mJ/pm². In another embodiment of the fifth aspect of the invention, the wavelength W of the incident electromagnetic radiation is the same when performing both steps(b.l) and (b.2). Advantageously, this simplifies the design, ensures the correct alignment of unshielding and reading irradiation beams and reduces the time lapse between unshielding and reading, thus increasing reading accuracy. In one embodiment, step (b.1) is performed with a radiation having a fluence F higher than the one of the radiation used in the reading step (b.2) and the wavelength W of the radiation used in both steps is the same.

In an alternative embodiment of the method of the fifth aspect of the invention, the wavelength of the incident electromagnetic radiation used in the step wherein the hole is created (b.1) is different from the incident electromagnetic radiation used in reading step (b.2).

In another embodiment of the method of the fifth aspect of the invention, the radiation used in the steps of creating the hole and reading the analyte, (b.1) and (b.2) has a wavelength, the same or different, comprised from 100 nm to 10 pm. The excitation wavelength and the plasmon resonance peaks may overlap or separate from each other.

In another embodiment of the method of the fifth aspect of the invention, the radiation in the steps of creating the hole and reading the analyte, (b.1) and (b.2), has a fluence, the same or different, equal or lower than 10 mJ/pm², from 0.01 to 1 mJ/pm², particularly from 0.01 to 0.5 mJ/pm²’

The wavelength W and fluence values will greatly depend on the material forming the electromagnetically active layer and the thermal energy (or temperature) required to break the sheathing layer and give rise to the hole. The wavelength “W’ of the radiation is the one absorbed by the electromagnetically active layer. When the electromagnetically active layer is a plasmonic layer, the wavelength “W’ of the radiation is the one at which the layer exhibits a localized surface plasmon resonance (LSPR). The skilled person can routinely find the appropriate wavelength and fluence values to perform the two-stage irradiation process.

In one embodiment of the method of the fifth aspect of the invention, the substrate comprises a gold-based particulate layer as electromagnetically active layer, and the radiation for performing the steps of creating the hole and reading the analyte, (b.1) and (b.2), have a wavelength W comprised from 100 nm and 1 pm, and a fluence from 0.01 to 1 mJ/pm². In one embodiment of method of the fifth aspect of the invention, the substrate comprises a gold-based particulate layer as electromagnetically active layer, and the radiation for performing the steps of creating the hole and reading the analyte, (b.1) and (b.2), have the same wavelength W comprised from 200 nm and 1 pm, but different fluences, comprised in the range from 0.01 to 1 mJ/pm² In another embodiment of method of the fifth aspect of the invention, the substrate comprises a gold-based particulate layer as electromagnetically active layer, and the radiation for performing the steps of creating the hole and reading the analyte, (b.1) and (b.2) have the same wavelength W, comprised from 400 nm and 900 pm, particularly from 500 nm to 800nm, but different fluences, such as the fluence of the radiation in the step of creating the hole, (b.1), is comprised from 0.07 to 1 mJ/pm², from 0.08 to 0.5 mJ/pm², or from 0.05 to 0.3 mJ/pm² and the fluence in the reading step, (b. 2), is comprised from 0.01 to 0.07 mJ/pm², particularly is selected from 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, and 0.07 mJ/pm². For instance, for the case of the examples provided below, wherein the electromagnetically active layer is a gold nanosphere-based layer, steps of creating the hole and reading the analyte were performed with a radiation having the same wavelength, 785 nm, but differing in the fluence (in b.1 of about 0.11 mJ/pm², whereas in b. 2. of about 0.03 mJ/pm²).

In another embodiment of the fifth aspect of the invention, the electromagnetic radiation irradiates the substrate through a lens, such as 5X, 10X, 20X, 50X, 100X. In another embodiment, the step of creating the hole, (b.1 )is performed by irradiating the substrate with the electromagnetic radiation through a lens. In this way, the size of the hole can be broadened. In another embodiment, the step of reading the analyte, (b.2) is performed by irradiating the substrate with the electromagnetic radiation through a lens. In another embodiment, both steps (creating of the hole+reading of the analyte) are performed by irradiating the substrate with the electromagnetic radiation through a lens.

In another embodiment of the fifth aspect of the invention, the steps of creating the hole (b.1) and reading the analyte(s) (b.2) are repeated in previously not irradiated areas of the substrate.

In another embodiment of the method of the fifth aspect of the invention, step (b.1) is performed with a beam of electromagnetic radiation. In another embodiment, steps (b.1) and (b.2) are performed with the same beam. This idea represents a useful technological solution oriented to solve this problem in SERS measurements, in a non-invasive, direct and fast manner, using a single laser and Raman setup. This process can be applied to a wide variety of applications in high-throughput screening and real time analysis.

In another embodiment of the method of the fifth aspect of the invention, the fluid FL is a gas or liquid sample, particularly a liquid sample, such as a bodily fluid, a flowing sample or a stagnant sample. Key indicators or parameters of the Raman signal include for example wavelengths and intensities of the emission peaks. The detection method of the present invention finds broad applications in areas including particle measurement, process control, and environmental monitoring.

The method of the present invention can be applied to the detection various types of analytes, substances or compositions for authenticity, concentration or integrity. In one embodiment, the method includes the following steps: depositing the sample to be detected onto the substrate; exposing the sample to the excitation wavelengths, sequentially or simultaneously; detecting the Raman signal of the sample; comparing the key indicators of the Raman signal with that of a reference signal or spectrum to determine the presence, amount, concentration or integrity of the composition.

The analyte to be tested is exposed to the laser source for a sufficient period of time to generate a Raman signal. In one embodiment, the period of time ranges from about 0.5 second to about 3 hours.

The analyte may be chemisorbed or physisorbed to the electromagnetically active surface. An analyte or substance is chemisorbed to the nanoparticles when chemical bonding is involved. For example, 4-mercaptobenzoic acid (4-MBA) may be immobilized through the chemical bonding of the thiol-moiety of 4-MBA to metal surface of the nanoparticles. An analyte or substance may also be physisorbed to the nanoparticles by means other than chemical bonding. In the example of crystal violet (CV), the molecules may be immobilized via its highly conjugated TT system which interacts with the nanoparticle surface via van der Waals forces. An analyte or substance may also be immobilized on the electromagnetically active layer by both chemisorption and physisorption. Although some examples of analytes are described in the example section, those skilled in the art would understand that the type of analyte or substance detected by the disclosed SERS substrate is not particularly limited and may also include others.

In order to enhance the detection of the Raman signal, the analyte or substance may also be functionalized to include various functional groups. The functional groups serve to immobilize the analyte, improve its stability, or provide other signal enhancement benefits. For example, an analyte or substance may be modified to incorporate a thiol group. Meanwhile, a reactive group may be shielded by known protecting groups (e.g. FMOC for amino group). Introduction of the appropriate groups can be determined by one of ordinary skill in the art without undue experiments.

The analyte to be tested may be disposed to the nanoparticles in various forms such as in the form of a solution or suspension. Another advantage of the detection method lies in the multiplexing ability of the nanoparticles. Two or more substances may be attached to the nanoparticles via chemisorption and/or physisorption simultaneously. For example, adenosine and hypoxanthine can be detected as a mixture, as shown below.

In the context of the present invention, an “analyte” refers to the molecule to be detected. Thus, the molecule can be a biological molecule (such as nucleic acids, amino acids, peptides, proteins, metabolites, and pathogens) or a chemical substance (for example contaminants).

In a sixth aspect the present invention provides a method for functionalizing the substrate of the invention. All the embodiments provided above, regarding the substrate, are also embodiments of the method of the sixth aspect of the invention.

Substrate functionalization techniques help in selective separation and/or recognition of target molecules.

In a first step of the method of the sixth aspect, the substrate is irradiated with an incident electromagnetic radiation of wavelength W, particularly with a beam of electromagnetic radiation, to create a hole in the sheathing layer. In one embodiment, the first step is performed by successively irradiating non-previously irradiated areas of the sheathing layer. In this embodiment a sheathing layer with several holes are obtained. All the embodiments provided above for step (b.1 ), regarding the incident electromagnetic radiation to produce the one or more holes in the sheathing layer, are also embodiments of this first step of the method of the sixth aspect of the invention.

In a second step of the method of the sixth aspect of the invention, the substrate is contacted with a reagent, and the reagent is immobilized on the area of the electromagnetically active layer exposed by the hole, thus obtaining the substrate functionalized with the reagent.

The immobilization of the reagent within the hole can be due to covalent or non-covalent interactions (electrostatic interactions, hydrogen bonds or hydrophobic interactions). The skilled person can routinely optimize the conditions of the second step to appropriately immobilized the reagent of interest, on the basis of the particular physico-chemical properties of the reagent, of the material forming the sheathing layer and, optionally, of the electromagnetically active layer.

In the context of the present invention, the “reagent” can be any entity which provides a particular application to the substrate of the invention. Illustrative non-limitative examples of “reagents” to be used in the context of the invention are antibodies, antigens, tags, nanoparticles, labelled and unlabelled, aptamers or detectable labels, among others. For example, immunoassays measure the concentration or the presence of a small molecule or a biomolecule in a solution by the use of an antigen or antibody, on the basis of the potential of the antibody to selectively identify and bind with a target antigen that separates the specific target antigen from components of the complex sample, reducing the interference by other irrelevant components. Therefore, the interaction between antibody-antigen is considered very strong and powerful due to the presence of electrostatic forces such as hydrophobic interactions, hydrogen bonds, van der Waals forces and ionic bonds. A blocking agent can also be used to prevent non-specific interactions between the reagent and the substrate.

Aptamer hybridization is a method in which a single-stranded ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecule makes a duplex by sequence-specific and non- covalent interactions. These nucleic acid ligands are engineered and depicted from oligonucleotide libraries through a method called “systematic evolution of ligands by exponential enrichment” (SELEX). By conjugating with the specific target molecule, these aptamers can either assimilate small molecules into their nucleic acid structure such as binding pockets or be involved in the structure of macromolecules. Therefore, these aptamers have been combined with different other optical detection methods for the quick detection of specific target analytes in a composite sample such as food. There are two types of chemical methods that are frequently used for combining nanomaterials with aptamers, namely non-covalent linkage and covalent linkage. Covalent linking includes a chemisorption process.

Another aspect of the invention provides a device or kit containing the above described composite. The device or kit may further include a light source which is able to produce wavelengths in the range of 100-1000 nm. In one embodiment, the light source produces wavelengths in the ranges of 150 nm to 900 nm or from 200 nm to 800 nm. In some embodiment, the light source produces one or more wavelengths, sequentially or simultaneously.

The device or kit may further include a detector for detecting the Raman signal of the analyte. Characteristics of the Raman signal include wavelength, range, number and intensity of peaks.

In some embodiments, the present invention provides kits and systems for use in monitoring the level of analytes in a bodily fluid. In some embodiments, the kits are kits for home use by a subject (e.g., a subject with diabetes). For example, in some embodiments, a sensor is implanted in the skin or the eye of a subject (e.g., by a medical professional) and the subject is provided with a device for monitoring levels of analyte (e.g., the subject places the device near the sensor and the device reads-out glucose levels). The subject can then use this information to maintain better control of blood glucose levels and avoid complications of the disease. In some embodiments, the sensor is used extra-corporeally by introducing a biological sample (e.g., blood) to the device.

In still further embodiments, the present device is used at home or by a medical professional to monitor exposure to pesticides (e.g., in agricultural workers). The workers receive a sensor and are then monitored using a detection device.

In yet other embodiments, the present invention provides systems comprising nanobiosensors and detection devices. For example, in some embodiments, the systems are combined with an insulin delivery device (e.g., an insulin pump) for use as an artificial pancreas. Such a device finds use in the treatment of individuals with diabetes who require regular insulin doses. In some embodiments, the detection device and pump are external (e.g., combined into one unit). The device takes readings from a sensor (e.g., implanted in the skin near the device), calculates blood glucose concentration, and administers an appropriate level of insulin. In other embodiments, the entire system is internal (e.g., implanted underneath the skin or located in the abdominal cavity). In some embodiments, the entire system is a single unit comprising a sensor, a detection device, and an insulin delivery device. On the other hand, in some embodiments, the systems of the present invention are directly incorporated into a reactor or fermenter to measure multi-analytes simultaneously and in real-time. Such manufactured devices support a biologically active environment where a chemical process is carried out by organisms or biochemically active substances derived from such organisms. These sensors are attached to the walls of the reactor tank and in contact with the liquid medium whereas the laser source is externally located. Such devices find use in measuring the metabolic activity inside bioreactors without disturbing the industrial process. These obtained metabolic parameters offers highly valuable information to control the manufacturing requirements.

Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word “comprise” encompasses the case of “consisting of”. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Reference signs related to drawings and placed in parentheses in a claim, are solely for attempting to increase the intelligibility of the claim, and shall not be construed as limiting the scope of the claim. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein. Examples

1. Materials:

• For the synthesis of gold nanoparticles

HAUCI4 3H2O (>99.9%, trace metal basis) was purchased from Alfa Aesar. Sodium borohydride (ReagentPlus®, >99%, NaBH4), cetyl-trimethyl-ammonium chloride (>98%, CTAC), L-Ascorbic acid (ACS reagents, >99%, AA), poly(ethylene glycol) methylether thiol average Mn 6000 (PEG-6K), sodium hypochlorite (6-14% active chlorine, Emplura®) were purchased from Sigma-Aldrich. All solutions, except HAuCk and CTAB, were prepared immediately before use. Purified Milli-Q water was used in all experiments (Millipore, 18.2 MQ cm). Glassware was cleaned with aqua regia and rinsed extensively with Milli-Q water before use.

• Poly lactide co-glycolide

Poly-lactide co-glycolide 75:25 25000 Mn Acid endcap,

Poly-lactide co-glycolide 85:15 50000 - 70000 Mn ester endcap was purchased from Sigma Aldrich.

Poly-lactide co-glycolide 95:5 25000 Mn Acid endcap was purchased from Polysciences.

• PDMS

Poly(dimethyl siloxane), PDMS sylgard 184 elastomer kit was bought from Sigma Aldrich.

• Raman analytes

4-Nitrothiophenol (96%, NTP) was purchased from Alfa Aesar, Adenosine (suitable for cell culture), 4- Mercaptobenzoic acid (90%, 4-MBA), Thiabendazole (> 99% powder), Crystal Violet (dye content > 90%), Nicotinamide (> 98% powder) and Hypoxanthine ( BioReagent) were purchased from Sigma-Aldrich.

Equipment:

Transmission electron microscopy (TEM) images were obtained with a JEOL JEM- 1400PLUS transmission electron microscope operating at an acceleration of 120kV.

UV-Vis-NIR spectra were measured with a Cary 5000 UV-Vis-NIR spectrophotometer.

Scanning electron microscopy (SEM) was performed using an environmental SEM (FEI Quanta 250). 20 kV or 5-20 kV acceleration voltages were used to take the images. A confocal Raman microscope (Renishaw inVia) equipped with a 1024 x 512 CCD detector, using a 785 nm laser excitation source. This system contains an optical microscope equipped with a motor-controlled stage. This optical set-up provides motorized XYZ locations of the samples, which allow a high positional accuracy. Control on the Z-axis enables users to focus the laser on the surface of the plasmonic substrate, whereas XY displacement governed the location of the laser beam over the substrate. Once the focus is made, on a specific area of the substrate selected by an optical image, a hole is created by laser irradiation at high irradiance. On the other hand, XY scanning allows for acquiring multiple SERS spectra at varying positions. This procedure can be used to generate a SERS map of the scanned area. SERS maps can be processed to detect areas of the substrate which were exposed to the solution and where the characteristic SERS fingerprint of the absorbed molecules can be recorded.

An AX5 infrared thermal camera (FLIR) was used to determine the temperature of the substrate during SERS measurements.

2.1. Synthesis and functionalization of gold nanoparticles

The synthesis of gold nanospheres was carried out by a process adapted from Hanske C. et a!., 2017.

First, small nanoparticles of 2 nm were prepared by adding 50 pL of a 0.05 M HAuCk solution to 5 mL of a 100 mM CTAC solution. Subsequently, 200 pL of a 0.02 M NaBFk solution (7.5 mg / 10 mL) was added under vigorous stirring. After 3 min, the mixture was diluted 10 times by a 100 mM solution of CTAC.

Then, the resulting nanoparticles were used as seeds to obtain 10 nm nanospheres by overgrowth. For this purpose, 900 pL of small seeds was added to a mixture of 40 pL of a 0.1 M solution of AA and 10 mL of a 25 mM solution of CTAC. Next, 50 pL of a 0.05 M HAuCk solution was added under vigorous stirring. The size of the particles was measured by TEM after depositing the dispersions on carbon-coated 400 square mesh copper TEM grids. It was confirmed that nanospheres having a size of about 10 nm were obtained. The UV-Vis absorbance spectra of the resulting 10 nm seeds showed a localized surface plasmon resonance (LSPR) centred at 520 nm. Then, the dispersion was left undisturbed for at least 1 h, and afterwards the nanospheres were centrifuged at 12000 rpm and washed at least 3 times with CTAC 25 mM to obtain more homogeneous particles.

For further overgrowth, 125 pL of 10 nm nanospheres was added to a solution containing 40 pL of a 0.1 M solution of AA and 10 mL of a 25 mM solution of CTAC. Subsequently, 50 pL of 0.05 M of HAuCkwas added to the mixture. Next, 10 pL of a diluted solution of sodium hypochlorite (from Sigma-Aldrich, having 1 to 1.5% of available chlorine), and 10 min later, 5 pL of a 0.05 M solution of HAuCk was added to the solution under continued stirring to remove the rough edges in the nanoobjects by oxidative etching. After 30 min, the final nanospheres were centrifuged at 4800 rpm for 15 min and redispersed in 500 pM CTAC. Afterward, the particles were then concentrated to ca. 5 mM Au° in a 500 pM solution of CTAC.

The size of the final particles was found to be 35 nm by TEM images.

Finally, the resulting gold nanospheres were functionalized with PEG ligand. To that end, 1 mg/mL of PEG6K was added to the concentrated sample of nanospheres and the mixture was stirred overnight at room temperature (RT). Excess unbound PEG was removed by 4-fold centrifugation at 4800 rpm for 15 min and redispersion of the sedimented NPs (35 nm) in CTAC 500 pM. The resulting nanoparticle dispersion was used in the following steps.

2.2. Assembly of gold nanosphere superlattices

A 2pL droplet of PEGylated gold nanoparticle dispersion (50 mM gold nanospheres of 35 nm calculated from the absorbance at 400 nm, 66% ethanol, 200 pM CTAC) was casted on a borosilicate microscope coverslip (MenzelTM, #1.5) as the target substrate. After 40 s waiting time, a nanostructured PDMS stamp was placed on top of the droplet. The PDMS stamp was manufactured using PDMS, Sylargd 184 (Dow Corning GmbH) by mixing at a 10:1 weight ratio the base and curing agent, following manufacturer’s instructions (for soft lithography, Dow Corning).

This template featured a square lattice of holes of 270 nm wide holes, with a spacing of 500 nm. The master for the manufacture of the template was obtained following Dore C. and colleagues (Dore C. et al., 2018). After 2 h and complete evaporation of the liquid, the PDMS template was carefully lifted off the borosilicate substrate. The resulting dried, nanostructured film consisted of a nanosphere superlattice representing the inverse structure of the template. The obtained superlattice contains nanoparticles periodically distributed in the X-Y plane, thus with a remarkable homogeneity. This high homogeneity was confirmed by SEM imaging

2.3. Immobilization of gold nanospheres on the support

Immobilization of the nanostructured film onto the glass support was achieved by an oxygen plasma process by 20 s, followed by a UV-ozone process (ProCleanerTM chamber) for 5 minutes. The oxygen plasma process was operated using a diener electronic nanoplasma cleaner at 100 W and 0.4 mbar oxygen pressure.

2.4. Coating fabrication Commercially available, solid PLGA 75:25 was dissolved in ethyl acetate by mechanically stirring PLGA granules in the chosen solvent for 2 hours. Stock solutions were kept in the fridge at 8°C, each vial was wrapped with parafilm to avoid solvent evaporation.

PLGA coatings were created by spin coating (Laurell WS-400B-6NPP LITE) a solution of the desired PLGA (weight percent, co-polymer ratio) on top of a nanoparticle superlattice.

To this end, 300 pL droplet of the solution was deposited on top of the superlattice such that it would wet the whole surface of the substrate. The spin coating process was then started at a speed of 1500 rpm for 30s. The thickness of the PLGA layer was measured to be 1.5 pm by SEM cross section analysis. In order to obtain a partially coated PLGA substrate, the same protocol was repeated using this time acetone instead of ethyl acetate.

2.5. Microfluidic chip

PDMS Sylgard® 184 was purchased from Sigma-Aldrich, microdevices were produced according to the methodology described by Shin Y. et a., 2012. Soft lithography was used to develop positive SU8 240-pm relief patterns with the desired geometry on a silicon wafer (Stanford University). Polydimethylsiloxane (PDMS, Sylargd 184, Dow Corning GmbH) was mixed at a 10:1 weight ratio of base to curing agent, following the manufacturer’s instructions (for soft lithography, Dow Corning).

The mixed solution was poured into the SU8 master and then degassed to remove air bubbles. Once the solution was cured, the replica-molded layer was trimmed, perforated and autoclaved. The PDMS device was then exposed to a plasma cleaning treatment (2 min) and subsequently bound to the PLGA-plasmonic substrate. Watertight adhesion to the plasmonic substrate was achieved by applying a soft pressure on the device with a sterile pair of tweezers. Flow was generated in the microfluidic channel by means of a Cetoni Nemesys syringe pump with the low-pressure module. The flow was set at 10000 pL/h and the outlet was connected to another syringe pumping at the same flow rate.

2.6. Silicone Chamber

To perform a controlled incubation of the selected analytes, a silicon chamber, which holds the liquid analyte solution, was 3D printed (see Fig. 7a).

This silicone chamber was prepared using an elastomer base silicone (Advanced Proser, AS 5702) loaded into a 10 mL clear syringe (PSY-E; Musashi Engineering, Ltd.) and printed with a diameter of 2 cm by a multiheaded 3D Discovery bioprinter (RegenHU, Switzerland) on a glass microslide (26 x 76 mm). In order to ensure that the total area occupied by the silicone chamber lies within the area of the plasmonic substrate employed in subsequent steps, the area of the internal space within the silicone chamber is smaller than the area of the plasmonic substrate.

2.7. Device for performing the SERS tests

The silicon chamber thus obtained was covered with the plasmonic substrate obtained as described in previous sections, thus creating an inner compartment wherein the analyte to be tested is added. The configuration of the chamber and the substrate was such that injection and replacement of the analyte solution could be performed via input and output channels. The gold nanoparticles-PLGA side of the plasmonic substrate was oriented toward said inner compartment, such that it is accessible to the analyte to be tested (Fig 7b). 3. Protocols

3.1. SERS analysis

SERS spectra were collected with a confocal Raman microscope (Renishaw inVia) equipped with a 1024 x 512 CCD detector, using a 785 nm laser excitation source. In those cases wherein the substrate was the one of the invention, the irradiance of the laser was regulated as follows: to create the hole in the PLGA layer the irradiance of the 785 nm laser was, as the lower limit, of 0.11 mW/pm² for 1 s through 50 x objective; the SERS signal of the diffusing analytes was usually recorded with 0.03 mW/pm² irradiance of 785 nm laser for 1 s. For comparative purposes, a control plasmonic substrate (with no PLGA coating) was also evaluated and, in that case, SERS spectra were collected with the 50x objective with 0.03 mW/pm² irradiance for 1s.

3.2. Measurement of the PLGA layer permeability

Here, permeability was defined as the capacity of the covering layer to prevent the analytes in solution from reaching the plasmonic substrate. To measure this term, a 20 pL drop of the analyte solution (either 100 pM adenosine or 100 pM 4-MBA solution) was deposited on the plasmonic substrate including the PLGA coating, and 100 SERS spectra were randomly acquired from different regions, with a low laser intensity (0.03 mW/pm²) at 785 nm. If the characteristic fingerprint of the analytes is not observed in the recorded SERS spectra, it can be concluded that the shielding layer (i.e. , the PLGA layer) is not permeable to the analytes in solution.

3.3. SERS multiplexing test

To confirm the spatial control at different times and analytes, the PLGA-coated plasmonic substrate was sequentially incubated with the following analytes at 100 pM concentration: 4-MBA (with an intense Raman peak around 1084 cm^-1), crystal violet (with an intense Raman peak at 1183 cm^-1), thiabendazole (with an intense Raman peak at 1015 cm^-1), and nicotinamide (with an intense Raman peak at 1032 cm^-1).

The test consisted of introducing 500 pL of the analyte solution in the device. When one analyte was added, a laser irradiance of 0.11 mW/pm² was applied for 1 second, thus creating a hole. The analyte was then removed from the silicone chamber, by aspiration with a micropipette, and after a cleaning step consisting of flowing water through the silicone chamber, the following analyte was injected. During this process of sequential incubations, a new hole was created in a different spot of the PLGA layer for each analyte. The distribution of holes along the X-axis was chosen to be with a step of 20 pm without changing their position in the Y-axis (with this step size, no overlap occurred between the created holes). Once all the different analytes were sequentially incubated and the corresponding PLGA-holes created, the whole area was mapped with a laser irradiance of 0.03 mW/pm², thus detecting the analytes retained in the plasmonic layer.

3.4. Bioreactor device and measurements of cell metabolic activity

In cellular experiments, a silicone chamber as the one obtained following protocol 2.6, using PLGA 95:5, was manufactured to obtain a biologically active cellular environment and its operability as a bioreactor was tested. To that end, HeLa cells (1x10⁶cel/mL) were laden inside the silicone chamber and the whole system was then assembled with the plasmonic substrate (obtained as described in previous sections), placing the gold nanoparticle covered with a PLGA layer 95:5 directly in contact with the extracellular milieu (see Fig. 7c).

Cells were cultured in cell media (Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) supplemented with 200 pM of adenosine. SERS measurements were recorded at 0 h and 24 h after metabolite addition to monitor changes in adenosine extracellular concentration along time. For this experiment, three holes were created at selected times over different locations of the PLGA coating. Irradiation with the 785 nm laser, at an irradiance of 0.11 mW/pm², for 5 s and through a 50x objective, ensured a complete degradation of the sheathing layer at the irradiated areas. Subsequently, SERS spectra were recorded at these positions by irradiating with the same 785 nm laser, and an irradiance of 0.03 mW/pm² for 10 s.

4. Results

4.1. SERS sensing with and without PLGA layer

A comparative gold NP substrate, lacking the sheathing layer, obtained following the same protocols as those described in sections 2.1 to 2.3, was incubated in 4-MBA by adding 20 pL of a 100 pM solution. Subsequently, the solution was removed, by micropipetting, and the substrate was rinsed with water for 30 s. In the following step, 20 pL of a 100 pM nicotinamide solution was added on top of the substrate. A further test was performed by replacing the nicotinamide solution with a mixture of 4- MBA+nicotinamide.

The SERS spectra after adding each analyte are shown in Fig. 1a. During incubation with 4-MBA, a characteristic MBA vibration at 1078 cm^-1 (solid line) was detected. However, after rinsing the substrate and incubating with nicotinamide, the peak of 4-MBA was still present in the SERS spectrum, while no peak associated to nicotinamide was observed, normally localized at the position marked by a dotted line in the spectra. From these data, it was concluded that 4-MBA could not be removed by rinsing the sample with water, and that the presence of 4-MBA blocked the binding of nicotinamide on the Au NPs. In the same way, when adding a mixture of 4-MBA + nicotinamide after 4-MBA incubation, no nicotinamide detection was achieved.

Then, the same test was performed but using the substrate of the invention, comprising the PLGA (75:25, 85:15 or 95:5) sheathing layer, all of the changes introduced in the solutions could be detected by SERS. Fig. 1 b shows the results obtained when the used PLGA was 75:25. It can be observed that, not only the characteristic peak of 4-MBA molecule around 1078 cm^-1 was detected, but also the nicotinamide peak at 1032 cm^-1. The same results were obtained when the PLGA used in the sheathing coating was 85:15 or 95:5.

As illustrated here, the substrate provided by the present invention overcomes the “memory effect”, frequently reported for plasmonic substrates, thereby allowing an efficient real time detection and even re-usability of SERS substrates.

This result indicates that the not-permeable sheathing layer applied on the plasmonic substrate initially blocks the interaction of analyte molecules with the sample. After performing the procedure described in 3.2, this behaviour of the PLGA layer acting as a barrier to prevent analyte diffusion, was consistently validated in all measuring points for the case of spin-coating from PLGA dissolved in ethyl acetate, due to the perfect coverage of the plasmonic substrate by the PLGA layer (see Fig. 2b, 2d). When the PLGA-coated substrate was not whole coating the plasmonic substrate, the results were not so good (see Fig. 2a, 2c).

PLGA is a thermodegradable polymer. As a consequence, the permeability of PLGA layers can be modified by heating, resulting in the interaction of the analyte molecules with the available plasmonic component.

In this set-up configuration, heat generated by plasmonic nanoparticles through light absorption and deexcitation of phonons is exploited to alter the permeability of the PLGA layer over the plasmonic substrates. By using a laser excitation with an elliptical spot size of 26x13 pm, an increase in local temperature leads to a precise degradation of PLGA in the irradiated area. Such a plasmonic heating was validated by means of infrared camera images. The obtained results showed a local temperature increase from 30 to 38 °C on the plasmonic superlattice, upon excitation with a 785 nm laser at 0.11 mW/pm². No temperature increase was detected if the laser light was focused outside of the plasmonic superlattice, which validated the hypothesis that heating was released from the gold nanoparticles and could not be obtained from the laser alone.

In this set of experiments, gold NP superlattices were chosen because they exhibit a high SERS signal due to the large enhancement of the electromagnetic field in the vicinity of self-assembled gold nanoparticles. Moreover, the homogeneous distribution of the nanoparticles over the sample, as confirmed by SEM images, allows for regular SERS signal on the whole surface of the sample. Notwithstanding, other plasmonic substrates can also fulfil this requisite, such as close-packed films of gold nanoparticles or single gold nanostars assembled with a high NP density. We should also mention here that other metallic particles, including Ag, Cu, Al, etc., as well as novel nanostructures made of dielectric materials have been used as SERS substrates due to their electromagnetic resonances, known as Mie resonances, and they are also potentially useful.

4.2. Hole Creation in the PLGA layer

The behaviour of the outer PLGA coating was studied through SERS and SEM analyses. To that end, the PLGA (either 75:25, 85:15 or 95:5)-coated plasmonic substrate of the invention was incubated with 20 pL of an adenosine solution 100 pM. First, the sample was irradiated with 0.03 mW/pm² for 1 s (Fig. 3a). Such a laser irradiation was not intense enough to give rise to the generation of a sufficiently efficient plasmonic heating to break the polymeric chains and create a hole in the PLGA layer. As a consequence, no trace of adenosine signal was observed in the recorded SERS spectra. Then, the sample was irradiated with 0.11 mW/pm² for 1 s, leaving a hole in the PLGA layer due to degradation of polymeric chains through plasmonic heating. By the time of hole creation, monitoring of SERS signal from adenosine was initiated (Fig. 3b), on account of molecular diffusion, through the thus generated hole, toward exposed gold nanoparticles. In a final step, the irradiance of the 785 nm laser was reduced again to 0.03 mW/pm², to record SERS from the opened hole. In this instance, the presence of adenosine was confirmed (Fig. 3c), displaying a SERS peak at 735 cm^-1.

A SERS map of the same region is provided in Fig. 3d, which revealed that the SERS signal due to adenosine was only visible in zone 2, where the hole was made in the PLGA layer, whereas zones 1 and 3, corresponding to parts of the substrate coated with PLGA, did not provide any signal. A lower laser irradiance was used during SERS scanning to avoid the unnecessary degradation of the PLGA layer, once the hole has been formed, and to map the areas of the plasmonic substrate in contact with the solution. The hole created in the PLGA layer by laser irradiation was imaged by SEM (20 kV of acceleration voltages), allowing to conclude that the hole had dimensions of few tens of micrometres (Fig. 3e). On the close-up image, Fig. 3f, nanoparticles without the coating layer can be seen in zone 2, whereas zones 1 and 3 present the sheathing layer, preventing the resolution of NP clusters. 4.3. SERS Multiplexing using high spatial control of PLGA degradation on plasmonic superlattices

As stated above, PLGA coating allows for the detection of different analytes using the same plasmonic substrate. This goal can be achieved on account of the micrometre scale control for PLGA degradation. As shown in Fig. 4, a precise control can be achieved in the generation and location of multiple holes in the PLGA film, including those located in close proximity. Such a control in hole location is possible because of the small spot size of the irradiation laser. The beam path can be focused on specific locations by using a moving stage and an optical microscope included in the Raman spectroscopy equipment. This feature of micrometre scale control for PLGA degradation allows us to create new holes at defined positions, every time SERS measurements are to be made for detection of changes in the solution under analysis.

SERS spectra corresponding to each region can be found below and above the SERS map in Fig. 4. The map was generated by using the SERS intensities at the wavenumbers of the characteristic peaks. Four regions display a meaningful signal on the SERS map, corresponding to the laser-irradiated regions. A high spatial control can thus be achieved between the 4 spots with a meaningful SERS signal.

We present 4 different SERS measurements on an area of 100x40 pm: which could translate into thousands of measurements on a substrate with an area of 1 cm². Not only a spatial control is achievable with the sheathing layer-coated substrate of the invention, but also a high capacity to detect a particular analyte in a given hole. As shown below ( Fig.

4). SERS spectra from each hole showed characteristic peaks from the analyte present at the time of laser irradiation. This is also indicative that the plasmonic substrate and the impermeable coating are stable enough to appropriately play their corresponding roles during consecutive holes and SERS mappings.

4.4. Real-time sensing using a microfluidic device

As a proof of concept of real-time sensing, a PLGA substrate was implemented with a microfluidic device attached on top of the PLGA layer. The whole microfluidic plasmonic device was mounted along with a syringe pump system, in order to control the flow of analytes in the chip, as described in previous section 2.7. SERS signals were measured through the glass layer. A scheme of the experiment can be found in Fig. 5. Using the measurement method previously described in 3.3., multiple hole positions and SERS measurements were used, but only for 4-MBA and thiabendazole as analytes, as well as pure water. It was found that the presence of analytes could be monitored in real-time within the microfluidic channel, on the basis of the most intense SERS peak from 4-MBA (at 1080 cm^-1) and thiabendazole (at 1014 cm^-1). Fig. 5 displays SERS spectra upon the sequential addition of 4-MBA, thiabendazole and water. As can be observed, the substrate of the invention responds differentially to the addition of each of the analytes (Fig. 5b).

The control experiment, using a substrate without PLGA layer, showed a SERS signal characteristic of the presence of 4-MBA upon its first addition, this signal being persistent upon addition of water and thiabendazole (Fig. 5a).

4.5. Real-time sensing of cell metabolic activity in bioreactors

The inventors of the present invention manufactured a bioreactor as disclosed in previous section 3.4, and added adenosine to reach a high extracellular concentration of 200 .M. The presence of different ectonucleotidase enzymes in the bioreactor caused a quick consumption of extracellular adenosine, converting it into hypoxanthine (Yegutkin G., 2008). Shown in Fig 6a are the SERS spectra of the pure metabolites.

In Fig. 6b, SERS spectra registered at different times (0 and 24 hours) are provided. As explained in method 3.4, SERS spectra were recorded at the selected times, on 3 different locations of the plasmonic substrate after having created the corresponding holes.

In these experiments, the covering PLGA layer allowed for accurately monitoring the conversion of adenosine to hypoxanthine by SERS. On the other hand, control assays without this protective PLGA layer could not sense this metabolic shift in real-time.

The cell supernatants for both experiments, control and PLGA devices, were collected and re-evaluated on fresh plasmonic sensors, which did not present interferences from previously adsorbed molecules (Fig. 6c). The obtained results validate PLGA-coated plasmonic substrates as a useful strategy to accurately measuring varying conditions inside a bioreactor over time.

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Clauses

For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

Clause 1. A layered substrate comprising,

(a) an electromagnetically active layer,

(b) a support layer adjacent to the electromagnetically active layer, and

(c) a thermolabile sheathing layer adjacent to the electromagnetically active layer, wherein, at least one of the layers adjacent to the electromagnetically active layer is transparent to an incident electromagnetic radiation of wavelength W; the sheathing layer:

- is not-permeable to a fluid FL; and

- is capable of being degraded at a temperature T; and the electromagnetically active layer: - is integrally attached to the support layer;

- is capable of converting electromagnetic energy carried by the incident electromagnetic radiation of wavelength W into thermal energy; and

- is thermostable at temperature T.

Clause 2. The substrate of clause 1, wherein the electromagnetically active layer produces the temperature T upon irradiation with an electromagnetic radiation of wavelength W.

Clause 3. The substrate of any one of the clauses 1-2, wherein the electromagnetically active layer produces the temperature T upon irradiation with an electromagnetic radiation of wavelength Wand fluence equal or above a fluence F.

Clause 4. The substrate of any one of the preceding clauses, wherein the electromagnetically active layer is capable of enhancing by a factor between 10^A1 and 10^A12 the intensity of the signal of at least one peak of the Raman scattering spectrum obtained for a target analyte contained in fluid FL in contact with the electromagnetically active layer, when irradiated with an incident electromagnetic radiation of wavelength W and irradiance below an irradiance P.

Clause 5. The substrate of any one of the preceding clauses, wherein the sheathing layer comprises one or more materials selected from the group consisting of polymers, blockcopolymers, proteins and other biomolecules, metal organic frameworks, covalent organic frameworks, organic molecular films and the like.

Clause 6. The substrate of clause 5, wherein the sheathing layer comprises polymeric and/or block-copolymer chains.

Clause 7. The substrate of clause 6, wherein the polymeric chains are selected from: polyacrylates (such as polyethyl, polybuthyl), polymetacrylates (such as poly methyl metacrylate), polyvinyl acetates, polyvinyl acetate copolymers (such as poly ethylene vinyl acetate), poly(lactic acid) polymers, and poly(lactic-co-glycolic acid) copolymers.

Clause 8. The substrate of any one of the clauses 6-7, wherein the polymeric chains are selected from the group consisting of: poly(lactic acid) polymers, poly(lactic-co-glycolic acid) copolymers, particularly poly(lactic-co-glycolic acid) copolymers, and combinations thereof.

Clause 9. The substrate of any one of the preceding clauses, wherein the sheathing layer has a thickness equal to or higher than the thickness of the electromagnetically active layer, particularly it has a thickness up to 10 pm, particularly from 0.5 to 10 pm, particularly from 0.8 to 2 pm.

Clause 10. The substrate of any one of the preceding clauses, wherein the sheathing layer is transparent to the electromagnetic radiation of wavelength W.

Clause 11. The substrate of any one of the preceding clauses, wherein the sheathing layer does not comprise plasmon particles.

Clause 12. The substrate of any one of the preceding clauses, wherein the electromagnetically active layer comprises one or more material selected from the group consisting of plasmonic metallic, dielectric, and semi-conductor materials.

Clause 13. The substrate of any one of the preceding clauses, wherein the electromagnetically active layer is in the form of a particulate layer or of a continuous layer, particularly in the form of a particulate layer.

Clause 14. The substrate of any one of the preceding clauses, wherein the electromagnetically active layer is a plasmonic layer.

Clause 15. The substrate of any one of the preceding clauses, wherein the thickness of the electromagnetically active layer is in the range from 1 nm to 300 nm, all subunits included.

Clause 16. The substrate of any one of the preceding clauses, wherein the electromagnetically active layer comprises at least one of the materials included in the group consisting of noble metals, transition metals, metal oxides such as noble and transition metal oxides, alkaline earth metal oxides, and combinations thereof.

Clause 17. The substrate of clause any one of the preceding clauses, wherein the electromagnetically active layer comprises a metal selected from the group consisting of silver (Ag), osmium (Os), palladium (Pd), platinum (Pt), gold (Au), rhodium (Rh), ruthenium (Ru), rhenium (Re), iridium (Ir) or any combinations or alloys thereof; a transition metals include iron (Fe), copper (Cu), nickel (Ni), zinc (Zn), cobalt (Co), manganese (Mn), chromium (Cr), molybdenum (Mo), tungsten (W), Aluminium, tin (Sn) and any combinations or alloys thereof.

Clause 18. The substrate of any one of the preceding clauses, wherein the electromagnetically active layer comprises gold.

Clause 19. The substrate of any one of the preceding clauses, wherein the electromagnetically active layer is a particulate layer comprising plasmonic nanoobjects. Clause 20. The substrate of any one of the preceding clauses, wherein the electromagnetically active layer is a particulate layer consisting of plasmonic nanoobjects homogenously distributed on the support.

Clause 21. The substrate of clause 20, wherein the plasmonic nanoobjects are selected from nanospheres, nanosheets, nanowires, nanofibers, and nanotubes.

Clause 22. The substrate of any one of the clauses 20-21 , wherein the plasmonic nanoobjects comprise a metal selected from the group consisting of Ag, Au, Cu, Al, Mg, Ni, Pd, Pt, alloys and combinations thereof.

Clause 23. The substrate of any one of the preceding clauses, wherein the plasmonic nanoobjects are surface functionalized, such as PEG-functionalized plasmonic nanoobjects.

Clause 24. The substrate of any one of the clauses 19-23, wherein the plasmonic nanoobjects are plasmonic aggregated nanoobjects.

Clause 25. The substrate of any one of the preceding clauses, wherein the electromagnetically active layer is a particulate plasmonic layer consisting of gold nanoobjects with a size from 5 to 100 nm, particularly from 10 to 50 nm, which are homogenously distributed on the support.

Clause 26. The substrate of clause 25, wherein the gold nanoobjects are gold nanospheres, particularly PEG-functionalized gold nanospheres.

Clause 27. The substrate of any one of the preceding clauses 1-13, wherein the electromagnetically active layer is made of a material selected from the group consisting of silicon, germanium, carbon, and/or lll-V semiconductor materials.

Clause 28. The substrate of any one of the preceding clauses, wherein the support layer is transparent to the electromagnetic radiation of wavelength W.

Clause 29. The substrate of any one of the preceding clauses, wherein the support layer is rigid or flexible.

Clause 30. The substrate of any one of the preceding clauses, wherein the electromagnetically active layer is directly and integrally attached to the support layer.

Clause 31. The substrate of any one of the preceding clauses, wherein the electromagnetically active layer is indirectly and integrally attached to the support layer by means of an interposed layer, particularly an interposed adhesive or metallic layer, such as a titanium layer. Clause 32. The substrate of any one of the preceding clauses, which further comprises an adhesive layer on the side of the support layer opposite to the side adjacent to the plasmonic layer.

Clause 33. The substrate of clause 32, wherein the adhesive layer comprises an adhesive material selected from the group consisting of polyethylenimine, polyvinyl acetate, phenol formaldehyde and ethylene vinyl acetate.

Clause 34. The substrate of any one of the preceding clauses, wherein the support layer comprises a material selected from the group consisting of a polymer, glass, quartz, ceramics, sapphire, and combinations thereof.

Clause 35. The substrate of clause 34, wherein the polymer is selected from the group consisting of silicone-based polymer (such as silicone, silicone nitride, and polydimethylsiloxane (PDMS)), polystyrene, polycarbonate, polyethylene, and combinations thereof.

Clause 36. The substrate of any one of the clauses 34-35, wherein the support comprises a silicon-based polymer, particularly the support consists of a silicon-based polymer.

Clause 37. The substrate of any one of the preceding clauses, which has a solid-planar conformation.

Clause 38. The substrate of any one of the preceding clauses, which is flexible.

Clause 39. The substrate of any one of the preceding clauses, wherein W is between 200 nm and 10 pm, particularly W is from NIR.

Clause 40. The substrate of any one of the preceding clauses, wherein F is below 10 mJ/m².

Clause 41 . The substrate of any of the preceding claims wherein fluence F is achieved by delivering an irradiance comprised from 10'⁶ mW/pm² and 10⁸ mW/pm² for an appropriate period of time.

Clause 42. The substrate of any one of the preceding clauses, wherein the fluid FL is a liquid.

Clause 43. The substrate of any one of the preceding clauses, wherein the fluid FL is a gas.

Clause 44. A process for the preparation of a layered substrate as defined in any one of the preceding clauses, the process comprising the steps of: (a) coating an electromagnet! cal ly active layer with a thermolabile sheathing layer; or, alternatively,

(ii) coating the resulting coated substrate of step (i) with a thermolabile sheathing layer, wherein the support layer, the electromagnetically active layer and the thermolabile sheathing layer are as defined in any of the clauses 1 to 43, wherein at least one of the electromagnetically active layer and the thermolabile sheathing layer is transparent to an incident electromagnetic radiation of wavelength W.

Clause 45. The process of clause 44, wherein the coating step (a) or (ii) is performed by a method selected from the group consisting of templated self-assembly, capillary selfassembly, droplet, electrostatic self-assembly, vapour deposition, sputtering, photo and electron lithography, laser writing and 3D-printing, and combinations thereof.

Clause 46. The process of clauses 44 to 45, wherein the coating of step (i) is performed by spin coating.

Clause 47. The process of clause 46 wherein the coating of step (i) is performed by a method selected from the group consisting of spin coating, dip coating, layer by layer assembly, interfacial self-assembly, sol-gel deposition, Langmuir-Blodgett, transfer, sputtering, electron beam lithography, soft lithography, nanoimprinting, nanosphere lithography and vapour deposition

Clause 48. The process of any one of the clauses 44 to 47, wherein when the electromagnetically active layer comprises metallic nanoobjects, then step (i) is performed by casting a metallic nanoobject dispersion on one side of the support layer.

Clause 49. The process of clause 48, wherein the casting comprises applying a droplet of the metallic nanoobject dispersion on the surface of the substrate and the subsequent application of a stamp on the droplet.

Clause 50. The process of any one of the clauses 44 to 49, which further comprises a step (1) comprising irradiating an area of the substrate resulting from step (a) or (ii) with an incident electromagnetic radiation of wavelength W, and, optionally, a step (2) wherein the electromagnetically active layer of the resulting substrate is functionalized with a reagent.

Clause 51. Use of the substrate as defined in any one of the clauses 1 to 43 to detect, quantify and/or monitor one or more target analyte(s) contained in a fluid FL. Clause 52. Use of the substrate as defined in any one of the clauses 1 to 43 as surface- enhanced Raman scattering (SERS) spectroscopy substrate.

Clause 53. Use of a material not-permeable to a fluid FL and capable of being degraded at a temperature T, to produce a sheathing layer as defined in any of the preceding clauses 1 to 11.

Clause 54. Use of a material which is not-permeable to a fluid FL and it is capable of undergoing thermal degradation at temperature T to confer spatio-temporal control in the detection, quantification and/or monitoring of one or more target analyte(s) to a layered substrate comprising an electromagnetically active layer adjacent to a support layer, wherein the material is in the form of a sheathing layer adjacent to the electromagnetically active layer, and the electromagnetically active layer, support layer and material forming the sheathing layer are as defined in any of the preceding clauses 1-43.

Clause 55. A method for identifying or quantifying one or more target analyte(s) contained in a fluid FL, the method comprising the steps of

(a) contacting a sample of the fluid FL with the sheathing layer of a substrate as defined in any one of the clauses 1 to 43; and

(b) obtaining a spectrum indicative of the amount or presence of the target analyte(s) in the sample.

Clause 56. The method of clause 55, wherein step (b) comprises the steps of:

(b.1 ) irradiating an area of the substrate as defined in any one of the clauses 1-43 with an incident electromagnetic radiation of wavelength W; and

(b.2) collecting a spectrum, such as a spectrum.

Clause 57. The method of clause 56, wherein the irradiating step (b.1) comprises the stages of: (b.1.1) generating the hole in the sheathing layer; and (b.1.2) reading the analyte in the electromagnetically active layer.

Clause 58. The method of any one of the clauses 56-57, wherein the fluence in the first stage (b.1.1) is higher than the fluence of the radiation in the second stage (b.1.2).

Clause 59. The method of any one of the clauses 57-58, wherein the fluence of the radiation in the first stage (b.1.1) is equal or above fluence F, whereas in the second stage (b.1.2) the fluence of the radiation is below fluence F.

Clause 60. The method of any one of the clauses 57-59, wherein stages (b.1.1) and (b.1.2) are repeated in non-previously irradiated areas of the substrate. Clause 61 . The method of any one of the clauses 55-60, wherein the electromagnetic radiation irradiates the substrate through a lens, such as 5X, 10X, 20X, 50X or 100X.

Clause 62. The method of any one of the clauses 55-61 , wherein step (b.1) is performed with a beam of electromagnetic radiation.

Clause 63. The method of any one of the clauses 55-62, wherein the wavelength of the incident electromagnetic radiation is the same when performing both stages (b.1.1) and (b.1.2).

Clause 64. The method of any one of the clauses 55-63, wherein the wavelength of the incident electromagnetic radiation used in stage (b.1.1) is different from the incident electromagnetic radiation used in stage (b.1.2).

Clause 65. The method of any one of the clauses 55-64, wherein W is between 200 nm and 10 pm, particularly W is from NIR.

Clause 66. The method of any one of the preceding clauses 55-65, wherein fluence F is achieved by delivering an irradiance comprised from 10'⁶ mW/pm² and 10⁸ mW/pm² for an appropriate period of time.

Clause 67. The method of any one of the clauses 55-66, wherein the fluid FL is a gas or a liquid sample, particularly a liquid sample, particularly a flowing sample or a stagnant sample.

Clause 68. A method for functionalizing the substrate, as defined in any one of the clauses 1 to 43, with a reagent, the method comprising a first step of creating one or more holes in the sheathing layer, and a second step of contacting the resulting substrate with a solution of the reagent.

Clause 69. The method of clause 68, wherein the creation of the hole comprises irradiating the substrate with an incident electromagnetic radiation of wavelength W, particularly with a beam of electromagnetic radiation.

Clause 70. The method of any one of the clauses 68-69, wherein the first step comprises successively irradiating non-previously irradiated areas of the substrate with an incident electromagnetic radiation of wavelength W, particularly with a beam of electromagnetic radiation.

Clause 71 . A substrate obtainable by the method of any one of the clauses 68-70.

Clause 72. A device or kit comprising a substrate as defined in any one of the clauses 1 to 43. Clause 73. The device of clause 72, which is selected from a reactor, an analytical equipment and a chip.

Clause 74. The device of any one of the clauses 72-73, which is capable of containing a fluid. Clause 75. The device of any one of the clauses 72-74, wherein the device is a cell vessel and the substrate is adhered to the inner wall of the vessel.

Clause 76. The device of any of the clauses 72-73, which is a medical device.

Claims

53 Claims

1. A layered substrate comprising,

(a) an electromagnetically active layer,

(b) a support layer adjacent to the electromagnetically active layer, and

- is not permeable to a fluid FL; and

- is integrally attached to the support layer;

- is thermostable at the temperature T at which the sheathing layer degrades.

2. The layered substrate of claim 1 , wherein the electromagnetically active layer is a plasmonic layer, particularly a particulate plasmonic layer comprising plasmonic nanoobjects.

3. The layered substrate of claim 2, wherein the plasmonic electromagnetically active layer is a particulate plasmonic layer comprising plasmonic nanoobjects, the plasmonic nanoobjects having one, two, three, four or all the following features:

(ii) are aggregated metal nanoobjects;

(iii) are the same or different and comprise one or more metals selected from the group consisting of silver (Ag), osmium (Os), palladium (Pd), platinum (Pt), gold (Au), rhodium (Rh), ruthenium (Ru), rhenium (Re), iridium (Ir), iron (Fe), copper (Cu), nickel (Ni), zinc 54

(Zn), cobalt (Co), manganese (Mn), chromium (Cr), molybdenum (Mo), tungsten (W), tin (Sn), alloys, and any combination thereof;

(iv) are surface functionalized, such as PEG-surface functionalized; and

(v) are homogenously distributed on support surface.

4. The layered substrate of any one of the preceding claims, wherein the sheathing layer is transparent to the electromagnetic radiation of wavelength W and comprises polymeric and/or block-copolymers chains; particularly polyacrylates (such as polyethyl, polybuthyl), polymetacrylates (such as poly methyl metacrylate), polyvinyl acetates, polyvinyl acetate copolymers (such as poly ethylene vinyl acetate), poly(lactic acid) polymers, poly(lactic- co-glycolic acid) copolymers, or any combination thereof.

5. The layered substrate of any one of the preceding claims, wherein the support layer is transparent to the incident electromagnetic radiation of wavelength W.

6. The layered substrate of any one of the preceding claims, which has a solid-planar conformation.

7. A process for the preparation of a layered substrate as defined in any one of the preceding claims, the process comprising the steps of:

(ii) coating the resulting coated substrate of step (i) with a thermolabile sheathing layer, wherein the electromagnetically active layer, the thermolabile sheathing layer, and the support are as defined in any one of the claims 1 to 7, and at least one of the electromagnetically active layer and the thermolabile sheathing layer is transparent to an incident electromagnetic radiation of wavelength W.

8. A method for functionalizing the layered substrate as defined in any one of the preceding claims 1-6 with a reagent, the method comprising the steps of creating one or more holes in the sheathing layer of the substrate, and contacting the resulting substrate with a solution comprising the reagent.

9. Use of the layered substrate as defined in any one of the claims 1 to 6 as a sensor to detect, quantify and/or monitor one or more target analyte(s) contained in a fluid FL by a spectroscopy-based technique.

10. Use of the layered substrate as defined in any one of the claims 1 to 6 as surface- enhanced Raman spectroscopy (SERS) substrate. 55

11. A method for identifying or quantifying one or more target analyte(s) contained in a fluid FL, the method comprising the steps of

(a) contacting a sample of the fluid FL with the sheathing layer of a layered substrate as defined in any one of the claims 1 to 7; and

(b.1) irradiating an area of the at least one of the transparent layers of the layered substrate with an incident electromagnetic radiation of wavelength W, particularly with a beam of electromagnetic radiation, to generate a hole in the sheathing layer; wherein the generation of the hole is performed by the electromagnetically active layer of the substrate which converts the electromagnetic energy carried by the incident electromagnetic radiation of the wavelength W, passing through the transparent layer(s), into thermal energy, to produce the temperature T at which the sheathing layer is locally degrades, thus producing the hole;

(b.2) irradiating the layered substrate resulting from step (b.1); and collecting the spectrum, particularly a SERS spectrum, indicative of the amount or presence of the target analyte(s) in the sample.

12. The method of claim 11 , wherein the fluence of the incident electromagnetic radiation during the step (b.1) is higher than the fluence of the incident electromagnetic radiation during step (b.2).

13. The method of claim 12, wherein the wavelength of the incident electromagnetic radiation is the same when performing steps (b.1) and (b.2).

14. The method of any one of the claims 11-13, wherein steps (b.1) and (b.2) are repeated in non-previously irradiated areas of the substrate.

15. A device for quantifying or detecting one or more analytes comprising a layered substrate as defined in any one of the claims 1 to 6.