WO2025003045A1

WO2025003045A1 - An improved sers substrate with a layer comprising halide

Info

Publication number: WO2025003045A1
Application number: PCT/EP2024/067628
Authority: WO
Inventors: Nakkiran Arulmozhi; Isidro BADILLO-RAMÍREZ; Anja Boisen
Original assignee: Danmarks Tekniske Universitet
Current assignee: Danmarks Tekniske Universitet
Priority date: 2023-06-30
Filing date: 2024-06-24
Publication date: 2025-01-02
Anticipated expiration: 2025-12-30

Abstract

A surface-enhanced Raman spectroscopy (SERS) substrate and methods for manufacturing SERS substrates is described. The SERS substrate comprises a gold surface portion on which a layer comprising a halide is provided. Described is a method for performing SERS for detection or sensing of an analyte, wherein the method comprises manufacturing a SERS substrate. Described is also a SERS substrate manufacturing kit suitable for manufacturing said SERS substrate and/or for performing SERS analyte detection and further the use of the provided SERS substrate(s) in a method for drug sensing.

Description

AN IMPROVED SERS SUBSTRATE WITH A LAYER COMPRISING HALIDE

TECHNICAL FIELD

The invention relates to a SERS substrate and methods for manufacturing SERS substrates. The invention also relates to a method for performing SERS for detection or sensing of an analyte, wherein the method comprises manufacturing a SERS substrate. The invention further relates to a SERS substrate manufacturing kit suitable for manufacturing said SERS substrate and/or for performing SERS analyte detection. Lastly, the invention relates to the use of the provided SERS substrate(s) in a method for drug sensing .

BACKGROUND

Surface-enhanced Raman spectroscopy (SERS) based sensors and assays have the advantage of combining label-free detection with ultrahigh sensitivity, allowing for rapid, non-invasive detection of target molecules by recording their molecular vibrational modes depicted in fingerprint spectrum. In this way, trace amounts of molecules can be detected. One application for such sensors or assays relates to therapeutic drug monitoring (TDM), where SERS is a powerful method for sensing relevant TDM molecules and thus provide insight to improve treatment procedures for various diseases (e.g . cancer).

The Raman measurement technique functions by directing monochromatic light provided by a light source, such as a laser, onto an analyte (or sample) that is to be chemically analysed (quantitatively detected). Most of the incident photons are elastically scattered by the analyte molecules. This means that the scattered photons have the same energy, and thus the same frequency, as the photons that were incident on the analyte. A small fraction of the photons (about 1 in 10⁷ photons) is inelastically scatted by the analyte molecules. These inelastically scattered photons is termed the "Raman effect". The inelastically scattered photons may have frequencies greater than, or typically, less than the frequency of the incident photons.

When an incident photon collides with a molecule, energy may be transferred from the photon to the molecule or from the molecule to the photon. When energy is transferred from the photon to the molecule, the scattered photon will emerge from the sample having a lower energy and a corresponding lower frequency. These lower-energy Raman scattered photons are commonly referred to in Raman spectroscopy as the "Stokes radiation". A small fraction of the analyte molecules is already in an energetically excited state. When an incident photon collides with an excited molecule, energy may be transferred from the molecule to the photon, which will emerge from the sample having a higher energy and a corresponding higher frequency. These higher-energy Raman scattered photons are commonly referred to in Raman spectroscopy as the "anti-Stokes radiation."

The Stokes radiation is detected by a detector, such as a photomultiplier or a wavelength- dispersive spectrometer, which converts the energy of the impinging photons into an electrical signal. The characteristics of the electrical signal are at least partially a function of the energy (or wavelength, frequency, wavenumber, etc.) of the impinging photons and the number of the impinging photons (intensity). The electrical signal generated by the detector can be used to produce a spectral graph of intensity as a function of frequency for the detected Raman signal (i.e., the Stokes). A unique Raman spectrum corresponding to the particular analyte may be obtained by plotting the frequency of the inelastica lly scattered Raman photons against the intensity thereof. This unique Raman spectrum may be used for many purposes such as identifying an analyte, identifying bonding of atoms and molecules in the analyte, and determining physical and chemical properties of the analyte. Raman spectroscopy may be used to analyse molecular species or mixtures of different molecular species. Furthermore, Raman spectroscopy may be performed on a number of different types of molecular configurations, such as organic and inorganic molecules in either crystalline or amorphous states.

Molecular Raman scattering of photons is a weak process. As a result, powerful, costly laser sources typically are used to generate high intensity excitation radiation to increase the weak Raman signal for detection. SERS is a technique that allows to enhance vibrational modes of a molecule by creating a stronger Raman signal from an analyte relative to conventional Raman spectroscopy. In SERS, the analyte molecules are adsorbed onto, or placed adjacent to, an activated nanostructured metal surface (a "SERS-active structure"). The interactions between the molecules and the nanostructured surface cause an increase in the strength of the Raman signal. The mechanism of Raman signal enhancement exhibited in SERS is not completely understood. Two main theories of enhancement mechanisms have been presented in the literature: electromagnetic enhancement and chemical (or "first layer") enhancement.

Several SERS-active structures have been employed in SERS techniques, including activated electrodes in electrolytic cells, activated metal colloid solutions, and activated metal substrates such as a roughened metal surface or metal "islands" formed on a substrate. For example, it has been shown that adsorbing analyte molecules onto or near a specially roughened metal surface made from gold (Au) or silver (Ag) may enhance the Raman scattering intensity by factors of between 10⁸ and IO¹⁰. SERS has also been performed employing randomly oriented nanometre scale needles, particles, and wires, as opposed to a simple roughened metallic surface. The intensity of the Raman scattered photons from a molecule adsorbed on such a metal surface may be increased by factors as high as 10¹⁴.

The type of metal surface also influences the sensitivity of the SERS surface. While nanostructured Au SERS substrates are chemically more stable and, therefore, are generally preferred for practical use, there are additional considerations for utilizing Ag metal: (i) the SERS signal enhancement using Au substrates is typically lower compared to Ag SERS substrates, and (ii) some analytes display higher affinity towards an Ag surface compared to Au. However, since Ag is susceptible to surface oxidation, this leads to significantly lower substrate stability, which is one of the major roadblocks in developing SERS-based assays. An optimal SERS sensor should sense only trace amounts of chemicals, thus there is a need for developing SERS substrates and methods for manufacturing these, to achieve increased sensitivity and stability of SERS substates.

SUMMARY

It has been found by the present inventor(s) that the sensitivity of a SERS substrate is improved if said substrate comprises a gold surface portion on which a layer comprising a halide is provide. The inventor(s) have further invented a method for the manufacture of a SERS substrate, a method for performing a SERS measurement for detection of an analyte and a SERS substrate manufacturing kit. In addition, the inventors provide a use of said SERS substrate manufacturing kit to obtain relevant SERS substrates and further provide a use of said SERS substrate in a method for drug sensing.

So, in a first aspect the present invention relates to a SERS substrate comprising a gold surface portion on which a layer comprising halide is provided.

In a second aspect the present invention relates to a method for the manufacture of a SERS substrate, the method comprising : providing a SERS substrate with a gold surface portion, applying, to the gold surface portion, an electrolyte solution comprising a halide ion source and depositing on the gold surface portion, by electrochemical deposition, a layer comprising halide.

In a third aspect the present invention relates to A method of performing a SERS measurement for detection or sensing of an analyte, wherein said method comprises: providing a SERS substrate with a gold surface portion, applying, to the gold surface portion, an electrolyte solution comprising the analyte and a halide ion source and simultaneously depositing on the gold surface portion, by electrochemical deposition, a layer comprising halide and performing the SERS measurement.

In a fourth aspect the present invention relates to a SERS substrate manufacturing kit, comprising a SERS substrate with a gold surface portion, a halide ion source and optionally a silver ion source.

In a fifth aspect the present invention relates to use of a SERS substrate manufacturing kit as disclosed, wherein said kit may be used to obtain a SERS substrate with a gold surface on which an electrochemically deposited layer comprising silver halide is provided.

In a sixth aspect the present invention relates to use of a SERS substrate, as disclosed, in a method for drug sensing, such as for sensing relevant therapeutic drug monitoring molecules i.e. analytes such as imatinib, busulfan and methotrexate, where an analyte to be tested is provided on the layer of the SERS substrate or in an electrolyte solution in contact with the layer of the SERS substrate and, wherein a SERS measurement is performed to sense the analyte

LEGENDS

Fig. 1 : Schematic illustration (Fig. lc) and SEM image (Fig Id) of a SERS substrate with a layer comprising halide as well as a schematic illustration (Fig. la) and SEM image (Fig. lb) of the reference SERS substrate. Also shown is a cyclic voltammograms provided from a SERS substrate with a layer comprising halide and the reference SERS substrate (Fig. le).

Fig. 2: Detection of busulfan using SERS substrate comprising halide. More specifically, EC- SERS of busulfan on an Au NP SERS substrate with (Fig. 2d-2e) and without Ag and halide ion co-deposition (Fig. 2a-2c).

Fig. 3: Detection of busulfan using an Au NP SERS substrate comprising halide. More specifically, EC-SERS band intensity at 1028 cm-1 as a function of busulfan concentration measured both with and without co-deposition of Ag and halide ions.

Fig. 4: Measurements at physiological pH (7.4). More specifically, SERS spectra for a busulfan concentration of 5 pM in an electrolyte solution comprising phosphate-buffered saline (PBS) solution. Fig. 5: Detection of methotrexate using SERS substrate comprising halide. More specifically, EC-SERS of methotrexate on SERS substrate with (Fig. 5d-5e) and without Ag and halide codeposition (Fig. 5a-5c).

Fig. 6: Detection of methotrexate using SERS substrate comprising halide. More specifically, the EC-SERS band intensity at 692 cm-1 as a function of methotrexate concentration measured both with and without co-deposition of Ag and halide ions.

Fig. 7: Schematic illustration of the setup and method for the manufacture of a SERS substrate. Fig. 8: XPS peak analysis of selected atomic species measured on the AuNP substrate comprising a layer comprising a halide, specifically Ag 3d (Fig. 8a) and Br 3d (Fig. 8b).

Fig. 9: XPS survey band analysis of the AuNP substrate comprising a layer comprising a halide.

Fig. 10 : Schematic illustration of the setup and method for performing a SERS measurement for detection or sensing of an analyte simultaneously depositing on the gold surface portion, by electrochemical deposition, a layer comprising the halide and performing the in-situ SERS measurement.

Fig. 11 : Repetition of detection of busulfan using SERS substrate comprising halide. More specifically, EC-SERS of busulfan on SERS substrate with Ag and halide co-deposition (Fig. lla-c).

Fig. 12: Calibration curves for EC-SERS of busulfan on SERS substrate with Ag and halide ion co-deposition.

Fig. 13: XPS peak analysis of selected atomic species measured on the AuNP substrate after EC-SERS of busulfan on SERS substrate with Ag and halide ion co-deposition, specifically Ag 3d (Fig. 13a) and Br 3d (Fig. 13b). Additionally, the intensity of Ag 3d peak, the Br3d peak and the Au 4f peak is provided (Fig. 13c).

Fig. 14: XPS survey analysis measured on the AuNP substrate after EC-SERS of busulfan on SERS substrate with Ag and halide ion co-deposition (Fig. 14a) as well as a composition depth profile analysis of the same substrate (Fig. 14b). Fig. 15: Further measurements at physiological pH (7.4). More specifically, SERS spectra for a busulfan in an electrolyte solution comprising phosphate-buffered saline (PBS) solution (Fig 15a-b) and in ultrafiltered human serum solution (Fig 15c).

Fig. 16: Reusability of the EC-SERS substrate. More specifically, SERS spectra obtained using EC-SERS and after cleaning cycles.

Fig. 17: Comparative example, AgNP with busulfan added to the substrate using drop casting.

DETAILED DISCLOSURE

SERS substrate with a layer comprising halide

In a first aspect, a SERS substrate comprising a gold surface portion on which a layer comprising halide is provided.

A SERS substrate refers to any substrate, which may be used for SERS measurements. Typically, such substrates comprise a nanostructured surface i.e. surfaces exposing metal nanoparticles, needles, wires, nanopillars etc. as such rough surfaces are suitable for enhancing a Raman scattering signal. The shape and size of the nanostructures on the surface, strongly affect the strength of the enhancement because these factors influence the ratio of absorption and scattering properties. Methods for manufacturing such SERS substrates are known in the art. On a SERS substrate a layer of gold may be added, if the substrate is not made of gold, such as by electron beam evaporation of gold. In this way, a SERS substrate comprising a gold surface portion may be provided.

In preferred embodiments of the SERS substrate, the gold surface portion defines a plurality of nanopillars. A plurality of gold nanopillars (AuNP) may be a dense stochastic array or ordered array of gold nanopillars. The nanopillars may be slender nanopillars having a taper angle of approximately 90° and an average diameter of 50-90 nm, such as 70 nm. The nanopillars preferably have, straight after the deposition, metal caps or portions at their extreme ends, such as exhibiting a mushroom-like morphology. Preferably these metal caps are mainly confined to the apex region of the nanopillars, thereby constituting an array of isolated plasmonic active metal particles. In this way, said substrate comprises a stochastic or a uniform and symmetric distribution of AuNPs. Preferably, the morphology of the substrate is homogeneous. Fabrication of conventional AuNP substrates is known in the art. The SERS substrate comprises a gold surface portion on which a layer comprising halide is provided. Herein, halide is used to refer to at least a first portion comprising a halogen and at least a second portion comprising at least one element or radical, such as an element or radical that is more electropositive than the halogen, wherein a first portion may be a halide ion and/or form part of a salt composition, wherein the second portion may be ions, form part of a complex formation and/or form part of a salt composition. The layer comprising the halide may comprise less than 0.25% halogen relative to the gold surface portion. The layer comprising the halide may comprise chlorine, bromine, iodine, preferably bromine.

The effect of halide is that 1) halide alters (change or form) the nanostructures on top of the gold surface portion hereby changing the surface electronic properties and with that improves the chemisorption properties of gold; 2) halide, optionally in the presence of additional positive ions, e.g. transition metal ions such as silver, copper or nickel, creates more effective clusters (by aggregation effects) between the analyte and the SERS substrate surface layer. In this way, the presence of a layer comprising halide on a SERS substrate comprising a gold surface portion may be an increased sensitivity in detection of an analyte.

The layer comprising halide may be a highly uniform and equal in thickness. Alternatively, the layer comprising halide may be characterized as islands or clusters. In preferred embodiments, the layer comprising halide comprises monolayer amount of halide. Herein, a monolayer amount of halide is defined as a thickness of one or a few monolayers such as less than five monolayers of halide. Preferably, the layer comprising the halide has a thickness of less than 2.0 nm such as less than 1.0 nm. Keeping the layer to comprise monolayer amount and/or a thickness of less than 2.0 nm such as less than 1.0 nm allows for an enhancement of the SERS signal as the plasmonic properties of gold provides a larger contributing to the SERS signal enhancing effect. Preferably, at least 10%, such as 20%, such as 30%, such as 50%, such as 90% of the surface of the SERS substrate comprises the halide layer.

As to the definition of halide, the layer comprising a halide may comprise or be a nanometric ionic layer, wherein said nanometric ionic layer is used to refer to a nanometer thick layer comprising such as essentially consisting of ions such as halide ions and ions of an element or radical that is more electropositive than the halogen. In this way, a nanometric layer may differ from a metallic layer, wherein electrons may move more freely. Having a nanometric ionic layer may provide the advantage that the layer comprising a halide (optionally further interacting with an analyte, see later paragraph) can be removed by applying voltage.

Having the layer comprising a halide essentially being a nanometric ionic layer provide a reusable substrate, where one substrate can be used multiple times analysing for an analyte from same origin (such as sample from one patient) such determining uncertainty of the analyte detection or one substrate can be used multiple times analysing for an analyte from a variety of origins (such as sample from multiple patients) hereby limiting waste and upscale analyte detection as the time spend or sample decrease (shorter handling time).

The layer comprising halide may further comprise silver and/or copper. Preferably, the layer comprising halide may further comprise silver. Hence, the layer comprising halide may comprise silver halide (i.e. comprises silver and halogen). The layer comprising silver halide may comprise or be a nanometric ionic layer comprising silver and halide ions. Additionally or alternatively, the layer comprising halide may comprise copper halide such as copper and chlorine, copper and bromine or copper and iodine. The layer comprising copper halide may comprise or be a nanometric ionic layer comprising copper and halide ions.

The layer comprising silver halide may comprise monolayer amount of silver halide. Again, monolayer amount of silver halide is defined as a thickness of one or a few monolayers such as less than five monolayers of silver halide. Preferably, the layer comprising silver halide has a thickness of less than 2.0 nm such as less than 1.0 nm. Keeping the layer to comprise monolayer amount and/or a thickness of less than 1.0 nm allows for an enhancement of the SERS signal as the gold surface and the silver may provide an additional enhancing effect by the dual or combined effect of the gold-silver substrate. In addition, the presence of silver further allows for an interaction for an analyte, which prefers to interact with silver compared to gold. Preferably, at least 10%, such as 20%, such as 30%, such as 50%, such as 90% of the surface of the SERS substrate comprises the silver halide.

The layer comprising the halide may comprise less than 0.25% halogen relative to the gold surface portion. Additionally, the layer comprising the halide may comprise less than 1.00% silver relative to the gold surface portion. Additionally or alternatively, wherein the layer comprising the halide comprises silver halide, the silver to halogen being in a ratio of between 1.5: 1 and 10: 1 such as 2: 1 to 6: 1 such as 3 :1 to 5: 1 such as 4:1. Having a layer comprising such silver to halogen ratio may allow for exploitation of beneficial silver-analyte interactions. Such provide a substrate with an increased sensitivity in detection of an analyte.

In embodiments, the layer comprising the halide may comprise silver and chlorine, silver and bromine, silver and iodine, preferably silver and bromine. In embodiments, the layer comprising the halide may comprise silver ions and chloride, silver ions and bromide, silver ions and iodide, preferably silver ions and bromide. In embodiments, the layer comprising silver halide may comprise silver chloride (AgCI), silver bromide (AgBr), and/or silver iodide (Agl), preferably silver bromide (AgBr).

Providing the layer comprising the halide, specifically a layer comprising silver halide reduces or prevents oxidation of the SERS substrate surface (exposed to the solution). Importantly, by a lack of oxidation should be understood that the exposed SERS substrate surface does not react with oxygen, at least to any significant degree, to form an oxidised layer. Typically, such an oxidised layer would result in a significant decrease in sensitivity of a surface comprising silver. Consequently, the provided substrate has the potential for significantly increasing the SERS sensitivity towards an analyte. This may be specifically relevant for sensing molecules that require therapeutic drug monitoring.

Method for the manufacture of a SERS substrate with a layer comprising halide

In another aspect, a method for the manufacture of a SERS substrate with a layer comprising halide is provided. More specifically, provided is a method for the manufacture of a SERS substrate, the method comprising : providing a SERS substrate with a gold surface portion, applying, to the gold surface portion, an electrolyte solution comprising a halide ion source and depositing on the gold surface portion, by electrochemical deposition, a layer comprising halide.

In this way, an additional aspect comprises a SERS substrate comprising a gold surface portion on which a layer comprising halide is provided by electrochemical deposition.

In embodiments, the SERS substrate with a gold surface portion may be as described above, such as preferably, the gold surface portion defines a plurality of nanopillars. The electrolyte solution comprising a halide ion source may refer to any solution comprising halide ions such as chloride, bromide and/or iodide. Preferably, said electrolyte solution further comprises a supporting electrolyte. In addition, the layer comprising a halide may preferably be as already disclosed such as may comprise monolayer amount of halide. Preferably, the layer comprising the halide may have a thickness of less than 2.0 nm, such as less than 1.0 nm. Preferably, the layer comprising halide may be a nanometric ionic layer. The layer comprising the halide may comprise less than 0.25% halogen relative to the gold surface portion. The layer comprising the halide may comprise chlorine, bromine, iodine, preferably bromine.

Electrochemical deposition refers to the migration of particles, such as ions, suspended in a liquid medium under the influence of an electric field followed by deposition of the particles onto an electrode, such as a working electrode, wherein the electrode is an electrically conductive surface such as a plasmonic material surface e.g. a gold surface. An electric field can be applied across a working electrode and a reference electrode by providing a voltage over said two electrodes, where the voltage is a quantitative expression of the potential difference between the two electrodes. Herein, in embodiments, the depositing step comprises, subsequent to providing the electrolyte solution, providing a voltage to the SERS substrate, between the SERS substrate as a working electrode, and a reference electrode. The electrodes function under wet conditions where the working electrode and the reference electrode are in contact with an electrolyte solution such that particles can migrate in the solution between the electrodes.

In preferred embodiments, the electrochemical deposition is achieved via underpotential deposition performed under wet conditions i.e. said substrate surface is in contact with an electrolyte solution. Underpotential deposition refers to electrodeposition of species at a potential less negative than the equilibrium potential for the reduction of said species. During underpotential deposition conditions, species such as an ion has a more energetically favoured interaction with the working electrode than the species-to-species interaction such as more favourable than particle-particle interaction in the crystal lattice of pure particles. However, a second layer of species on the working electrode may be less likely to be energetically favoured, wherefore said underpotential deposition allows for monolayer amount such to control the thickness to be on the order of monolayer of atoms i.e. monolayer amount on said SERS gold surface. Consequently, said method may provide a thickness of less than 2.0 nm such as less than 1.0 nm.

As the voltage applied relates to a potential difference between the reference electrode and the SERS substrate functioning as the working electrode, achieving underpotential deposition conditions depends on the choice of reference electrode. In preferred embodiments, the working electrode is a gold electrode as the SERS substrate comprises a gold surface portion. In preferred embodiments, the reference electrode is an Ag/AgCI reference electrode. The choice of reference electrode defines a threshold value (i.e. potential of zero charge) for when particles of positive charge (below threshold) or negative charge (above threshold) will be deposited on the working electrode. The threshold value for an Ag/AgCI reference electrode is 0.4 V, when relative to the working electrode comprising a gold surface portion. In preferred embodiments, the voltage is above a threshold voltage during a deposition time interval of 180-800 s. In further preferred embodiments, the voltage applied is in the range of 1.3 V to 0.4 V. In this way, negative ions such as halide ions will be moved to the working electrode i.e. the SERS substrate.

In a specifically preferred embodiment of the method for manufacturing a SERS substrate, the SERS substrate comprises a gold surface portion, which defines a plurality of nanopillars, the reference electrode is an Ag/AgCI reference electrode, and the voltage applied is in the range of 1.3 V to 0.4 V.

As disclosed, the deposition occurs in an electrolyte solution comprising a halide ion source. The electrolyte solution comprising a halide ion source may refer to any solution comprising halide ions. A valid halide ion source comprises silver halides such as silver chloride (AgCI), silver bromide (AgBr), and/or silver iodide (Agl), preferably silver bromide (AgBr). In preferred embodiments, the electrolyte solution comprises a concentration of halide ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM. Keeping the concentration below 1.0 pM allow for the system to avoid silver halide precipitation. Typically, silver halides are silver chloride (AgCI), silver bromide (AgBr), and/or silver iodide (Agl). Other halide ion sources comprise copper halides or nickel halides, wherein said copper halides may be cobber chloride, cobber bromide and/or cobber iodide.

Preferably, the solution further comprises a supporting electrolyte. The electrolyte solution may be an acidic solution, such as a perchloric acid (HCIO4) solution, a sulfuric acid solution, or a hydrochloric acid solution; or a buffered solution, such as a phosphate-buffered saline (PBS) solution; or a human serum solution such as an ultrafiltered human serum. The ultrafiltered human serum may be prepared by ultra filtration using filters with a cutoff value of 3, 10, 30 kD, preferably 10 kD. Most preferably, the electrolyte solution is acidic as halide ions are active in acidic media. The choice of electrolyte may depend on choice of biomedical application. A buffered solution such as a phosphate-buffered saline (PBS) solution or an ultrafiltered human serum solution may be preferred, when resembling physiological relevant conditions. Additionally, when resembling physiological conditions, the pH of the electrolyte solution may preferably be between 7.0-8.0, such as 7.0-7.8, such as of 7.2-7.6, such as 7.4.

Method for the manufacture of a SERS substrate with a layer comprising silver halide

The method for the manufacture of a SERS substrate may further comprise that the electrolyte solution additionally comprises a silver ion source and wherein the deposition step comprises depositing, on the gold surface portion, by electrochemical deposition, a layer comprising silver halide. In this way, an additional aspect comprises a SERS substrate comprising a gold surface portion on which a layer comprising silver halide is provided by electrochemical deposition. The layer comprising halide may further comprise less than 1.00% silver relative to the gold surface portion. Specifically, the layer comprising silver halide, said silver halide comprises halogen selected from chlorine, bromine and/or iodine, preferably wherein the halogen is bromine. In embodiments, the silver halide comprises silver chloride (AgCI), silver bromide (AgBr), and/or silver iodide (Agl), preferably silver bromide (AgBr). The layer comprising halide may comprise silver halide, wherein the silver to halogen may be in a ratio of between 1.5: 1 and 10: 1 such as 2: 1 to 6: 1 such as 3: 1 to 5: 1 such as 4: 1. Additionally, the layer comprising halide such as silver halide may further comprise copper.

Specifically, the method may further comprise: a. subsequent to applying, to the gold surface portion, the electrolyte solution; providing a first voltage to the SERS substrate, wherein said first voltage is either above or below a threshold voltage. b. subsequent to step a. providing a second voltage to the SERS substrate, wherein if said first voltage is above the threshold voltage, said second voltage is below the threshold voltage or if said first voltage is below the threshold voltage said second voltage is above the threshold voltage.

Optionally, the method may further comprise a cleaning step such to clean the gold surface portion of the SERS substrate prior to halide deposition. The cleaning of the SERS substrate is enabled by the method further comprising; applying, to the gold surface portion, at least one cycle of cyclic voltammetry to the SERS substrate with a gold surface portion prior to halide deposition. At least one cycle of cyclic voltammetry is used to refer to cycling the potential of a working electrode. Specifically, a cycle of cyclic voltammetry may comprise changing the potential to include a lowest potential and a highest potential using a scan rate, said scan rate defining the size of the steps for potential change. The lowest potential may be -1.0 V and the highest potential may be 2.0 V. Preferably, a cycle of cyclic voltammetry may comprise the lowest potential equal to or above -0.6 V and the highest potential of 1.3 V, such as comprise the lowest potential of -0.4 V and the highest potential of 1.3 V. Preferably, the scan rate is 0.5 V/s in a range potential range from -0.4 V to 1.3 V. Specifically, the cycle may be conducted by first increasing the potential from 0.0 V to 1.3 V, followed by decreasing the potential to -0.6 V, followed by increasing the potential to 0.1 V. Preferably, the at least one cycle may be at least 3 cycles such as may be a minimum of 5 cycles.

Providing a first voltage to the SERS substrate results in migration of either particles of positive charge (if the applied voltage is below threshold) or negative charge (if the applied voltage is above threshold) to the surface of the working electrode. Providing the second voltage results in migration of particles of opposite charge, compared to the particles which were attracted applying the first voltage. While applying the second voltage, a portion of the particles which migrated to or near the surface of the working electrode while providing the first voltage, will stay at the surface of the working electrode whereas a second portion may migrate towards the reference electrode. However, as a result of thermodynamics, the positive and negative ions at the working electrode surface deposit as a layer on the SERS substrate surface. Hence, a layer comprising both positive and negative charge is deposited on the working electrode, i.e. the SERS substrate. In this way, in embodiments, said method results in deposition of a layer comprising a silver halide on the gold surface portion of the SERS substrate. The specifically provided deposition step may again exploit the underpotential deposition mechanism. In this way, said method may provide a SERS substrate comprising a gold surface on which a layer comprising a silver halide is provided, wherein said layer comprises monolayer amount of silver halide. In some embodiments, said layer comprising monolayer amount of silver halide has a thickness of less than 2.0 nm, such as less than 1.0 nm.

In a specifically preferred embodiment: the reference electrode is an Ag/AgCI reference electrode, the first voltage is in the interval 0.4 to 1.3 V and is applied in a deposition time interval of 160-200 s, and the second voltage is in the interval of -0.8 to 0.2 V and is applied in a deposition time interval of 180-800 s.

This preferred embodiment comprises an Ag/AgCI reference electrode and said Ag/AgCI reference electrode has a threshold value of 0.4 V (i.e. potential of zero charge), when relative to the working electrode comprising a gold surface portion. Hence, this embodiment results in a first migration of at least a portion of the halide ions to the SERS substrate followed by a second migration of at least a portion of the silver ions and subsequent in the deposition of a layer comprising silver halide. Immediate switching from the first voltage to the second voltage may result in a higher coverage of the layer comprising silver halide on said gold surface of the SERS substrate. Immediate switching may be defined as the switch occurring within a second or less than a second. This is because ions may leave the surface when the surface approaches an electrochemical potential of zero, which is given by the threshold value. The effect of ions leaving the surface is kept low by the immediate switch.

In an alternative specific embodiment, the reference electrode is an Ag/AgCI reference electrode, the first voltage is in the interval -0.8 to 0.2 V and is applied in a deposition time interval of 160-200 s and the second voltage is in the interval 0.4 to 1.3 V of and is applied in a deposition time interval of 180-800 s.

Equivalent to the previous embodiment, this embodiment result in a first migration of at least a portion of the silver ions to the SERS substrate followed by a second migration of at least a portion of the halide ions and subsequent in the deposition of a layer comprising silver halide. Again, immediate switching from the first to the second voltage is preferred for embodiments as an immediate switch may result in a higher coverage of the layer comprising silver halide on said gold surface of the SERS substrate. Similarly, to the specific embodiment above, it is preferred to reduce the effect of ions, migrated to the SERS substrate surface during the application of the first voltage, leaving the surface prior to being deposited as a silver halide layer.

The electrolyte solution may comprise a concentration of halide ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM. In The electrolyte solution may further comprise a concentration of silver ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM. A suitable halide and silver ion source comprise silver halides such as silver chloride (AgCI), silver bromide (AgBr), and/or silver iodide (Agl), preferably silver bromide (AgBr). Keeping concentration of silver ions and halide ions below 1.0 pM allow for the system to avoid silver halide precipitation. Silver halides are a preferred source for halide and silver ions as no further ions or compounds need to be introduced into the manufacture of the SERS substrate such that no additional potential contamination is introduced to said SERS substrate originating from the source of the ions. Other halide ion sources comprise copper halides or nickel halides. Another silver ion source comprises AgNOs or AgCI Preferably, the electrolyte solution further comprises a supporting electrolyte. The electrolyte solution may be an acidic solution, such as a perchloric acid (HCIO4) solution, a sulfuric acid solution, or a hydrochloric acid solution; or a buffered solution such as a phosphate-buffered saline (PBS) solution; or a human serum solution such as an ultrafiltered human serum. The ultrafiltered human serum may be prepared by ultra filtration using filters with a cutoff value of 3, 10, 30 kD, preferably 10 kD. Most preferably, the electrolyte solution is acidic as halide ions are active in acidic media. The choice of electrolyte may depend on choice of biomedical application. A buffered solution such as a phosphate-buffered saline (PBS) solution or an ultrafiltered human serum solution may be preferred, when resembling physiological relevant conditions. Additionally, when resembling physiological conditions, the pH of the electrolyte solution may preferably be between 7.0- 8.0, such as 7.0-7.8, such as of 7.2-7.6, such as 7.4.

In a specifically preferred embodiment, the SERS substrate comprises a gold surface portion, which defines a plurality of nanopillars, the reference electrode is an Ag/AgCI reference electrode, the solution comprises silver ions, bromide ions and an electrolyte, the first voltage applied is in the interval 0.4 to 1.3 V, the second voltage is in the interval of -0.8 to 0.2 V.

Embodiment results in a SERS substrate comprising a gold surface portion on which a layer comprising silver halide, is deposited, preferably wherein said layer comprises monolayer amount of silver halide, preferably silver bromide.

The method for the manufacture of a SERS substrate with a layer comprising halide such as silver halide, provide a method for providing a SERS substrate suitable for detection or sensing of an analyte by performing a SERS measurement. Said layer comprising halide has the potential for significantly increasing the sensitivity toward an analyte in a SERS measurement. This may be specifically relevant for sensing molecules that require therapeutic drug monitoring.

Method of performing a SERS measurement for detection or sensing of an analyte

In one aspect, a method of performing a SERS measurement for detection or sensing of an analyte, wherein said method comprises: providing a SERS substrate with a gold surface portion, applying, to the gold surface portion, an electrolyte solution comprising the analyte and a halide ion source and simultaneously depositing on the gold surface portion, by electrochemical deposition, a layer comprising halide and performing the SERS measurement is also provided.

An analyte may be defined as any chemical molecule (such as a molecule of < 10 kD molecules, such as a molecule of <5 kD molecules) which identification or concentration in a solution may be of interest. The analyte may preferably be ionic or polarizable in the electrolyte solution (i.e. aqueous solutions).

The method of performing a SERS measurement for detection or sensing of an analyte may comprise applying, to the SERS substrate, a voltage above or below a threshold value while performing the SERS measurement i.e. the detection or sensing may be achieved during an electrochemical SERS (EC-SERS) measurement. Hence, co-deposition of ions from the solution may occur while performing the SERS measurement. In this way, the method of simultaneously depositing on the gold surface portion, by electrochemical deposition, a layer comprising the halide and performing the SERS measurement, may be achieved by simultaneous co-deposition of ions such as at least co-deposition of one ion species from the solution resulting in a SERS substrate comprising a gold surface portion on which a layer comprising halide is provided while performing the SERS measurement. The SERS measurement comprises receiving radiation from the substate. The forming of the layer may comprise providing a voltage to the substrate to attract ions thereto, and the simultaneity may comprise providing the voltage at the same time as receiving radiation from the substrate. Clearly, the providing of the voltage may be initiated before or after initiation of the receipt of radiation from the substrate, and these processes need not end at the same point in time either. In this context, as is also seen above and further below, the halide may comprise a first and a second portion, and these portions may be provided on the substrate simultaneously or staggered in time, such as by providing different voltages (often with different polarities) to the substrate over time as described below. The analyte may engage or attach to the layer comprising the halide after this layer has been formed or during its formation. Alternatively, the analyte may engage or attach to one of the first or second portions already before forming part of the layer.

Preferably, where the SERS measurement is performed applying a voltage above a threshold value, negatively charged particles such as halide ions, preferably bromide, migrate to the surface during the SERS measurement. The SERS measurement may be performed applying only a first voltage above a threshold value or the SERS measurement may be performed applying both a second voltage above a threshold value and a first voltage below a threshold value. In this way, halide co-deposition may take place during the SERS measurement. Within these embodiments, preferred analytes comprise analytes which are negatively charged under the solution conditions applied or alternatively are polarized. In this way, an increased presence of analytes on the SERS surface during measurement can be achieved wherefore an increased sensitivity of the vibrational modes of the analyte may be achieved.

Preferably, where the SERS measurement is performed by applying a voltage below a threshold value, positively charged particles, such as silver ions, migrate to the surface during the SERS measurement. The SERS measurement may be performed applying a second voltage below a threshold value, wherein a first voltage applied was above a threshold value. In this way, Ag co-deposition may take place while measuring. Additionally or alternatively, where the SERS measurement is performed by applying a voltage below a threshold value, positively charged particles, such as silver ions and/or copper ions, migrate to the surface during the SERS measurement. The SERS measurement may be performed applying a second voltage below a threshold value, wherein a first voltage applied was above a threshold value. In this way, Ag and/or Cu co-deposition may take place while measuring. Within these embodiments, preferred analytes comprise analytes which are positively charged under the solution conditions applied. Similarly, to the previous embodiments, an increased presence of analytes on the SERS surface during measurement can be achieved wherefore an increased sensitivity of the vibrational modes of the analyte can be achieved. Then, very low amounts of said analyte may be detected enabling detection limits such as below 1 pM, such as below 0.5 pM, such as of 0.1 pM.

Also, specifically relevant for SERS measurements performed applying a second voltage below a threshold value, while having halide and silver ions in solution provide for preferred analytes result in an enhanced SERS signal. Preferred analytes comprise analytes, which have strong coordination of silver ions such as analytes comprising a sulfonate domain (e.g. sulfonic acid, methanesulfonate, mesylate), wherein the oxygen of the sulfonate domain coordinates with said Ag ions during the Ag co-deposition or after formation on top of the Au surface. Furthermore, the induced deformation in the O=S=O or -S(=O)2 domain, during the applied potential, might also contribute simultaneously to the analyte attraction to the surface and, therefore, to the SERS signal relating to the C-C backbone band enhancement. Hence, the presence of silver ions might influence the analyte molecular adsorption and orientation, which may also be a contributing factor to an enhanced SERS signal.

As of the above, provided is a method of performing a SERS measurement for detection or sensing of an analyte, wherein said method comprises: providing a SERS substrate with a gold surface portion, applying, to the gold surface portion, an electrolyte solution comprising a halide ion source and the analyte and simultaneously depositing on the gold surface portion, by electrochemical deposition, a layer comprising halide and performing the SERS measurement.

The method of performing a SERS measurement may further comprise that the electrolyte solution further comprises a silver ion source, that said method further comprises simultaneously depositing on the gold surface portion, by electrochemical deposition, a layer comprising silver halide and performing the SERS measurement.

Additionally, or alternaively, the method of performing a SERS measurement may further comprises an electrolyte solution comprising a copper ion source, that said method further comprises simultaneously depositing on the gold surface portion, by electrochemical deposition, a layer comprising copper halide and performing the SERS measurement.

Preferably, the SERS measurement such as the EC-SERS mapping is conducted simultaneously with or immediately after initiating the second potential. The term "immediately after" may be defined as the initiation of the EC-SERS mapping occurring within less than five seconds such as within a second or less than a second after initiating the second potential. Consequently, simultaneous deposition on the gold surface portion of a layer comprising the halide and performing the SERS measurement is achieved.

The method may comprise that i) the reference electrode is an Ag/AgCI reference electrode, ii) the first voltage is in the interval 0.4 to 1.3 V and is applied in a deposition time interval of 160-200 s, and iii) the second voltage is in the interval of -0.8 to 0.2 V and is applied in a deposition time interval of 180-800 s.

Alternatively, the method may comprise that i) the reference electrode is an Ag/AgCI reference electrode, ii) the first voltage is in the interval -0.8 to 0.2 V and is applied in a deposition time interval of 160-200 s, and iii) the second voltage is in the interval 0.4 to 1.3 V of and is applied in a deposition time interval of 180-800 s.

It is specifically preferred, that the method further comprises that the layer comprising halide comprises chlorine, bromine, iodine, preferably bromine. Specifically, the method may further comprise, wherein the layer comprising silver halide, the silver halide comprises silver chloride (AgCI), silver bromide (AgBr), and/or silver iodide (Agl), preferably silver bromide (AgBr). It is also preferred that the halide ion source and the layer comprising halide comprises the same halogen. Preferably, the layer comprising halide may be a nanometric ionic layer.

The method may further comprise that the electrolyte solution comprises a concentration of halide ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM. Additionally, wherein the electrolyte solution comprises a concentration of silver ions, the concentration may be equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM. The method may further comprises that said electrolyte solution is an acidic solution, such as a perchloric acid (HCIO4) solution, a sulfuric acid solution, or a hydrochloric acid solution; or a buffered solution, such as a phosphate-buffered saline (PBS) solution; or a human serum solution such as an ultrafiltered human serum.

The method may further specify, that the gold surface portion defines a plurality of nanopillars.

Method of performing a SERS measurement for detection or sensing of an analyte - cleaning and optionally a second SERS measurement

Of specifical interest, the method may further comprise a cleaning step which may enable reusability of the SERS substrate with a gold surface portion after a SERS measurement. The reusability of the SERS substrate is enabled by the method further comprising; applying, to the gold surface portion, at least one cycle of cyclic voltammetry to the SERS substrate with a gold surface portion after a SERS measurement. At least one cycle of cyclic voltammetry is used to refer to cycling the potential of a working electrode. Specifically, a cycle of cyclic voltammetry may comprise changing the potential to include a lowest potential and a highest potential using a scan rate, said scan rate defining the size of the steps for potential change. Preferably, a cycle of cyclic voltammetry comprises the lowest potential equal to or above - 1.0 such as equal to or above -0.6 V and the highest potential of below 2.0 V such as of 1.3 V, such as comprise the lowest potential of -0.4 V and the highest potential of 1.3 V. Preferably, the scan rate is 0.5 V/s in a range potential range from -0,4 V to 1,3 V.

Specifically, one cycle may be conducted by first increasing the potential from 0.0 V to 1.3 V, followed by decreasing the potential to -0.6 V, followed by increasing the potential to 0.1 V. Preferably, the at least one cycle may be at least 3 cycles such as may be a minimum of 5 cycles. Consequently, the method may further comprise a cleaning step, applying, to the gold surface portion, at least one cycle of cyclic voltammetry to the SERS substrate with a gold surface portion after a SERS measurement.

The method comprising a cleaning step may further comprise exchanging the electrolyte solution applied, to the gold surface portion, to a cleaning electrolyte solution prior to at least one cycle of cyclic voltammetry. The cleaning electrolyte solution is used to refer to an electrolyte solution not comprising the second analyte. Preferably, the cleaning electrolyte solution consists of an acidic solution, such as a perchloric acid (HCIO4) solution, a sulfuric acid solution, or a hydrochloric acid solution; or a buffered solution, such as a phosphate- buffered saline (PBS) solution.

Alternatively or additionally to the method comprising a cleaning step, the method may further comprise performing a second SERS measurement for detection or sensing of a second analyte, wherein said method comprises the SERS substrate with a gold surface portion after a SERS measurement. This method step specifically allows for reusing the SERS substrate with the gold surface portion. Specifically, a second SERS measurement may be obtained from a second electrolyte solution comprising the same analyte, wherein the analyte may be originated from the same sample source (e.g. the same sample from a patient) or from a different sample source (e.g. sample obtained from the same patient at a different time, or sample from a different patient). Consequently, a second analyte may be the same analyte as for any previous SERS measurement(s) or a different analyte.

More specifically, the method may further comprise performing a second SERS measurement for detection or sensing of a second analyte, wherein said method comprises the SERS substrate with a gold surface portion after a SERS measurement, applying, to the gold surface portion, at least one cycle of cyclic voltammetry, further applying, to the gold surface portion, a second electrolyte solution comprising a silver ion source, a halide ion source and the second analyte and simultaneously depositing on the gold surface portion, by electrochemical deposition, a layer comprising halide and performing the second SERS measurement. Preferably the halide ion source and the silver ion source may comprise silver chloride (AgCI), silver bromide (AgBr), and/or silver iodide (Agl), preferably silver bromide (AgBr). Hence, in these embodiments, said ion sources are both desired. This may prevent the presence of other, undesirable molecules/compounds in the electrolyte solution.

The method comprising performing a second SERS measurement may further comprise that the second electrolyte solution further comprises a concentration of silver ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM and/or a concentration of halide ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM. The second electrolyte solution may be an acidic solution, such as a perchloric acid (HCIO4) solution, a sulfuric acid solution, or a hydrochloric acid solution; or a buffered solution, such as a phosphate-buffered saline (PBS) solution; or a human serum solution such as an ultrafiltered human serum. The ultrafiltered human serum may be prepared by ultra filtration using filters with a cutoff value of 3, 10, 30 kD, preferably 10 kD. Most preferably, the electrolyte solution is acidic as halide ions are active in acidic media. The choice of electrolyte may depend on choice of biomedical application. A buffered solution such as a phosphate-buffered saline (PBS) solution or an ultrafiltered human serum solution may be preferred, when resembling physiological relevant conditions. Additionally, when resembling physiological conditions, the pH of the electrolyte solution may preferably be between 7.0-8.0, such as 7.0-7.8, such as of 7.2-7.6, such as 7.4.

The method comprising performing a second SERS measurement may further comprise exchanging the electrolyte solution applied, to the gold surface portion, to a cleaning electrolyte solution prior to at least one cycle of cyclic voltammetry. Preferably, the cleaning electrolyte solution comprises such as consists of an acidic solution, such as a perchloric acid (HCIO4) solution, a sulfuric acid solution, or a hydrochloric acid solution; or a buffered solution, such as a phosphate-buffered saline (PBS) solution.

SERS substrate manufacturina kit

In another aspect, a SERS substrate manufacturing kit is provided, said kit comprising a SERS substrate with a gold surface portion, a halide ion source and optionally a silver ion source.

The gold surface portion of the SERS substrate may define a plurality of nanopillars.

The halide ion source and optionally the silver ion source may comprise silver chloride (AgCI), silver bromide (AgBr), and/or silver iodide (Agl), preferably silver bromide (AgBr). Hence, said ion sources are both desired. This may prevent the need for other, undesirable molecules/compounds in the ion sources in the SERS substrate manufacturing kit.

The halide ion source may comprise copper halide. Specifically, the halide ion source may comprise a silver halide and a copper halide. The copper halide may be copper chloride, silver chloride, and/or copper iodide.

The SERS substrate manufacturing kit may also comprise one or more electrolyte solution(s) such as one or more acidic solution(s) or buffered acidic solution(s). More specifically, the electrolyte solutions may be an acidic solution, such as a perchloric acid (HCIO4) solution, a sulfuric acid solution, or a hydrochloric acid solution; or a buffered solution, such as a phosphate-buffered saline (PBS) solution; or a human serum solution such as an ultrafiltered human serum. Specifically, the electrolyte solution comprising the electrolyte, the electrolyte may be perchloric acid, sulfuric acid, hydrochloric acid, and/or phosphate such as in a concentration of 0.05-0.15M such as 0.1M.

The buffered solution such as a phosphate-buffered saline (PBS) solution or an ultrafiltered human serum solution may be preferred, for resembling physiological relevant conditions. The pH of the electrolyte solution being a buffered solution, or an ultrafiltered human serum solution may preferably be between 7.0-8.0, such as 7.0-7.8, such as of 7.2-7.6, such as 7.4

Preferably, the one or more electrolyte solution(s) comprises i) the halide ion source and/or ii) the silver ion source. Specifically, the electrolyte solution may comprise a concentration of halide ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM. Additionally or alternatively, the electrolyte solution may comprise a concentration of silver ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM.

In a specific embodiment, the SERS substrate manufacturing kit comprises a SERS substrate with a gold surface portion, wherein said gold surface portion defines a plurality of nanopillars, and further comprises an electrolyte solution comprising dissolved silver bromide (AgBr).

An additional aspect relates to the use of a SERS substrate manufacturing kit as described above, wherein said kit is used to obtain a SERS substrate with a gold surface on which an electrochemically deposited layer comprising silver halide is provided.

Use of a SERS substrate for drug sensing One aspect relates to the use of a SERS substrate as described above in a method for drug sensing, such as for sensing relevant therapeutic drug monitoring molecules i.e. analytes such as busulfan, imatinib and methotrexate, where an analyte to be tested is provided on the layer of the SERS substrate or in an electrolyte solution in contact with the layer of the SERS substrate, and wherein a SERS measurement is performed to sense the analyte.

Preferred analytes relevant for drug sensing comprise busulfan, imatinib or methotrexate. Busulfan is a bifunctional alkylating antineoplastic agent commonly used in chemotherapybased conditioning before hematopoietic cell transplantation and for treating chronic myelogenousleukemia, polycythemia vera and some types of blood cancers. Alternatively, imatinib and methotrexate are widely used anticancer drugs for several types of cancer, such as leukemia, breast cancer, gastro-intestinal stromal tumours, and lymphoma.

Above, the SERS substrate is described having a gold surface. Another aspect of the invention relates to a SERS substrate with a surface portion of any plasmonic material. Hence, a SERS substrate comprising a plasmonic material surface portion is provided on which the above-described layer comprising a halide is provided. In other words, an additional aspect comprises a SERS substrate comprising a plasmonic material surface portion on which a layer comprising a halide is provided by electrochemical deposition. Yet another aspect comprises a SERS substrate comprising a plasmonic material surface portion on which a layer comprising a silver halide is provided by electrochemical deposition.

A plasmonic material is a metal or a metal-like material that exhibits negative real permittivity. The most common plasmonic materials are gold, silver or copper. However, many other materials show metal-like optical properties in specific wavelength ranges. In this way, the plasmonic material may be a plasmonic metal comprising gold, silver and/or copper. The plasmonic material may also be an alloy preferably comprising gold or silver such as Fe- Ag; Ag-Ti, Ag-Cu or Si-Ag having the desired plasmonic material properties. Alternatively, the plasmonic material may be plasmonic-magnetic materials such as plasmonic-magnetic silica. As explained above, the plasmonic material surface portion may have a variety of shapes and structures, preferably the plasmonic material surface portion defines a plurality of nanopillars. The plurality of plasmonic nanopillars may be a dense stochastic array or ordered array of nanopillars. Preferably, the morphology of the substrate is homogeneous.

As mentioned above, the halide layer preferably is a monolayer amount of halide. Additionally or alternatively, a SERS substrate comprising a plasmonic material surface portion on which a layer comprising a halide is preferred, where the halide layer has a thickness of less than 1.0 nm. In preferred embodiments, the SERS substrate comprises a plasmonic material surface portion on which a layer comprising a silver halide is provided. The layer comprising silver halide may be a monolayer amount of silver halide. The layer comprising silver halide may comprise silver chloride (AgCI), silver bromide (AgBr), and/or silver iodide (Agl), preferably silver bromide (AgBr).

For all embodiments disclosed herein relating to the method for the manufacture of a SERS substrate, the SERS substrate may be any SERS substrate comprising a plasmonic material surface portion. Hence, said method may comprise: providing a SERS substrate with a plasmonic material surface portion, applying, to the plasmonic material surface portion, a electrolyte solution comprising a halide ion source and depositing on the gold surface portion, by electrochemical deposition, a layer comprising the halide.

Similarly, for all embodiments disclosed herein relating to a method of performing a SERS measurement for detection or sensing of an analyte, the SERS substrate may be any SERS substrate comprising a plasmonic material surface portion. Hence, a method of performing a SERS measurement for detection or sensing of an analyte, wherein said method comprises: providing a SERS substrate with a plasmonic material surface portion, applying, to the plasmonic material surface portion, an electrolyte solution comprising a halide ion source and the analyte and simultaneously depositing on the gold surface portion, by electrochemical deposition, a layer comprising the halide and performing the SERS measurement is also provided.

Equivalently, for all embodiments disclosed herein relating to the SERS substrate manufacturing kit, the SERS substrate may be any SERS substrate comprising a plasmonic material surface portion. Hence, a SERS substrate manufacturing kit is provided, said kit comprising a SERS substrate with a plasmonic material surface portion, a halide ion source and optionally a silver ion source.

For all embodiments relating to the use of a SERS substrate manufacturing kit or substrate as disclosed herein, the SERS substrate may be any SERS substrate comprising a plasmonic material surface portion. In this way, one aspect of the invention further comprises: use of a SERS substrate manufacturing kit as provided in any embodiments of the SERS substrate manufacturing kit where the SERS substrate comprises a plasmonic material surface portion, wherein said kit may be used to obtain a SERS substrate with a plasmonic material surface portion on which an electrochemically deposited layer comprising silver halide is provided. Additionally, one aspect of the invention further comprises: use of a SERS substrate according to any one of the SERS substrates disclosed, where the SERS substrate comprises a plasmonic material surface portion, in a method for drug sensing, such as for sensing relevant therapeutic drug monitoring molecules i.e. analytes such as busulfan, imatinib and methotrexate, where an analyte to be tested is provided on the layer of the SERS substrate or in an electrolyte solution in contact with the layer of the SERS substrate and, wherein a SERS measurement is performed to sense the analyte.

EXAMPLE 1

SERS substrate with a layer comprising a halide

This example serves to compare the gold nanopillar (AuNP) substrate before and after halide layer deposition (schematically illustrated in Fig. la and lc).

The following two substrates were used as references: i) AuNP substrate where no potential had been applied and ii) an AuNP substrate to which a 0.1 V potential was applied with the supporting electrolyte of HCIO4 in a 0.1 M concentration in the absence of halide and Ag ions. Fig. la shows a schematic illustration of the reference SERS substrate(s).

The SERS substrate with a layer of silver bromide (AgBr) was manufactured in an Ag ioncontaining and Br ion-containing 0.1 M perchloric acid (HCIO4) solution by electrochemical deposition on the AuNP substrate applying an initial voltage of 0.9 V (vs. Ag/AgCI) for 180 s followed by immediate change from 0.9 V to 0.1 V and applying 0.1 V for 420 s. Fig. lc shows a schematic illustration of the SERS substrate with a layer of silver bromide (AgBr).

The SEM image of the SERS substrate with a layer of silver bromide (Fig. Id) shows a gradual change in the structure and order of the NPs, more specifically a tendency to create clusters of NP (reference Fig. lb). This morphological change suggests that AgBr is coated on top of the AuNP or induces an aggregation effect i.e. a leaning effect on the NPs.

Additionally, the AgBr electrochemical deposition was identified through a cyclic voltammogram (CV) (Figure le), in which the AuNP substrate is the working electrode and acts as the cell for the reduction and oxidation of molecular species in the electrolyte. The CV was performed using a maximum voltage of 1.3 V (high E), minimum voltage of -0.6 V (low E) and a scan rate of 0.5 V/s. The CV of a bare AuNP substrate, recorded in the presence of HCIO4 solution, is depicted by the black line, showing a typical Au-surface oxidation and reduction profile. The grey line indicates the CV of Au after Ag [0.5 pM] and Br [0.5 pM] deposition in HCIO4, where the typical Au oxidation peaks are less visible, and the surface oxidation and the current reduction increase, attributed to the presence of deposited AgBr. In this way, a layer of silver bromide has formed on the SERS substrate. We note that for all the following experiments, the SERS substrates used are AuNP substrates.

EXAMPLE 2

EC-SERS of busulfan on SERS substrate with and without Ag and halide ion co-deposition.

The optimal analyte detection in acidic solution was performed in three steps: i) cleaning of the AuNP substrate by placing 500 pL of HCIO4 [0.1 M] without the analyte and applying five cycles of cyclic voltammetry using -0.6 V as the lowest potential, 1.3 V as the highest potential and a scan rate of 0.5 V/s; ii) preconditioning of the substrate, with the aim to reduce the hydrophobic property of the AuNP substrate, which was performed by replacing the HCIO4 cleaning solution with the HCIO4 electrolyte solution (500 pL [0.1 M]) containing the analyte, including or in the absence of Ag and Br ions, then the voltage is reduced to a first voltage of 0.9 V, in 0.2 V increment steps, to the 0.9 V which is applied for 180 s; iii) EC-SERS mapping was performed by the immediate change from 0.9 V to 0.1 V and simultaneous EC-SERS mapping along 420 s of continuously applied potential. The AgBr codeposition was simultaneously performed under the same EC-SERS parameters, by adding a solution of AgBr to reach a final concentration of Ag ions and Br ions of 0.5 pM. The concentration of analyte, here busulfan, was between 0 and 2.0 pM. All measurements were performed in triplicates.

Fig. 2a shows the average SERS spectra of busulfan solutions at different concentrations on the AuNP substrate when applying a constant potential of 0.1 V for 420 s, in the absence of halide and silver ions. It is observed that the main vibrational band of busulfan at 1028 cm-1 is rather weak at 0.5 pM and becomes gradually more intense when increasing the concentration of busulfan in the solution. Moreover, additional characteristic bands of busulfan at 820, 1176 and 1212 cm-1 are also defined at busulfan concentrations higher than 5 pM.

Fig. 2b shows an EC-SERS map of busulfan band distribution (intensity as a function of wavenumber, where the wavenumber range specifically relates to the highest intense band of the busulfan, the 1028 cm-1 band) along the applied potential from 180 to 600 s, using a busulfan concentration of 1.0 pM. Fig. 2c shows the intensity distribution as a function of wavenumber in the SERS map along the applied potential time of 420 s (the time provided in time frames i.e. zones such that zone 1 corresponds to time 180 s and zone 11 to 600 s), wherein the insert provides the intensity variation of the 1028 cm-1 along the SERS mapping. Considering the SERS band profile along the time of potential application and the band intensity of the main peak at 1028 cm-1, a favourable band enhancement occurs at the beginning of the potential application time, but has decreased to around half of maximum intensity after around half of the potential application time (inset Fig. 2c).

Fig. 2d shows the average SERS spectra of busulfan solutions at different concentrations using electrochemical co-deposition with Ag and halide ions on the AuNP substrate, when applying a constant potential of 0.1 V for 420 s. A clear and intense band at 1028 cm-1 is visible at 0.5 pM, and it becomes more intense with increasing busulfan concentrations. At the busulfan solution concentration of 10 pM, the main busulfan band intensity is enhanced around 8 times when comparing with the EC-SERS without Ag and halide ion co-deposition (Fig. 2a).

Fig. 2e shows an EC-SERS map of busulfan band distribution (intensity as a function of wavenumber, where the wavenumber range specifically relate to the highest intense band of the busulfan, the 1028 cm-1 band) along the applied potential from 180 to 600 s, using a busulfan concentration of 1.0 pM, and Ag and halide ion co-deposition. Fig. 2f shows the intensity distribution as a function of wavenumber in the EC-SERS map along the applied potential time of 420 s (the time provided in time frames i.e. zones such that zone 1 correspond to time 180 s and zone 11 to 600 s), wherein the insert provides the intensity variation of the 1028 cm-1 along the map. The main busulfan intensity band profile increases with the time of applying a constant potential. The main busulfan band is observed intense from the beginning of the measurement but the highest intensity, doubled the value, is observed at the medium-end part of the map (see Fig 2f insert). Furthermore, the increase in band intensity is also observed for the other busulfan related bands at 820, 859, 1176 and 1212 cm-1.

Fig. 3 shows the EC-SERS band intensity at 1028 cm-1 as a function of busulfan concentration in an electrolyte solution comprising 0.1 M HCIC measured both with and without co-deposition of Ag and halide ions, when applying a constant potential of 0.1 V for 420 s (so at time 600 s). The busulfan detection is efficient already in a linear range between the concentration range between 0.5 to 10 pM. However, in the Ag and halide co-deposition experiment a clear performance improvement is observed. Under the Ag and halide codeposition conditions, the busulfan detection is saturated at 5 pM and a detection below of 0.5 pM is enabled (Fig 3 insert, sigmoidal fitting). In this way, this illustrates that busulfan can be detected down to concentrations of between 2-20 pM.

In this way, the Ag and halide ion co-deposition causes a significant effect by enhancing the busulfan vibrational modes under the EC-SERS experimental conditions, allowing detection limits of a lower molecular concentration. EXAMPLE 3

Measurements at physiological pH

Fig. 4 shows average SERS spectra for a busulfan concentration of 5 pM in an electrolyte solution comprising phosphate-buffered saline (PBS) solution (pH 7.4), when applying a constant potential of 0.1 V. The SERS experiment was performed with and without Ag and halide ion co-deposition.

Fig. 4 demonstrates that the band at 1028 cm-1 is still the most evident and, again, highly intense in the system containing Ag and halide ions. However, in this case, the band intensity is slightly lower in comparison with the HCIO4 electrolyte media (Fig 2a, Fig. 3). This might be a consequence of the presence of additional salts in the electrolyte media and due to the drastic change in the pH of the solution. Nevertheless, the developed method can be applied at a physiological pH value, which might significantly expand the applications of EC-SERS and Ag and halide co-deposition for sensitive and reproducible detection.

EXAMPLE 4:

EC-SERS of methotrexate on SERS substrate with and without Ag and halide ion codeposition.

To explore the sensitivity of the developed substrate and the applicability of the developed method to other relevant drugs for TDM, we investigated the detection of methotrexate. All experimental parameters were equivalent to Example 2, only replacing busulfan with methotrexate.

Fig. 5a shows the average SERS spectra of methotrexate solutions at different concentrations on the AuNP substrate when applying a constant potential of 0.1 V for 420 s, in the absence of halide and silver ions. It is observed that some characteristic vibrational bands of methotrexate are slightly noticed when methotrexate is in a concentration higher than 0.5 pM. The characteristic bands become gradually more intense when increasing the concentration of methotrexate in the electrolyte solution. Specifically, the characteristic bands at 1558, 1359, 968 and 698 cm-1 are well defined and become intense at 2 pM methotrexate.

Fig. 5b shows an EC-SERS map of methotrexate band distribution (intensity as a function of wavenumber, where the wavenumber range is specifically related to the highest intense band of the methotrexate, the 698 cm-1 band) along the applied potential from 180 to 600 s, using a methotrexate concentration of 1.0 pM. Fig. 5c shows the intensity distribution as a function of wavenumber in the SERS map along the applied potential time of 420 s, (the time provided in time frames i.e. zones such that zone 1 correspond to time 180 s and zone 12 to 600 s) wherein the insert provides the intensity variation of the 698 cm-1 along the SERS mapping. Considering the SERS band profile along the time of potential application and the band intensity of the main peak at 698 cm-1, a favourable band enhancement occurs at the beginning of the potential application time and the intensity of the band decreases considerably as a function of potential application time (inset Fig. 5c).

Fig. 5d shows the average SERS spectra of methotrexate solutions at different concentrations using electrochemical co-deposition with Ag and halide ions on the AuNP substrate, when applying a constant potential of 0.1 V for 420 s. Methotrexate bands are visible from 0.25 pM, and most of the characteristic bands are defined at 0.5 pM. All of the characteristic bands become more intense with increasing methotrexate concentrations, specifically the characteristic bands at 1577, 1461, 1396, 1359, 1205 and 698 cm-1 are significantly enhanced. Additionally, the characteristic bands are seen to slightly shift (a certain wavenumber positions) in comparison to the methotrexate SERS band profiles obtained without co-deposition with Ag and halide ions.

Fig. 5e shows an EC-SERS map of methotrexate band distribution (intensity as a function of wavenumber, where the wavenumber range is specifically related to the highest intense band of the methotrexate, the 698 cm-1 band) along the applied potential from 180 to 600 s, using a methotrexate concentration of 1.0 pM and Ag and halide ion co-deposition. Fig. 5f shows the methotrexate intensity distribution in the EC-SERS map along the applied potential time of 420 s (the time provided in time frames i.e. zones such that zone 1 correspond to time 180 s and zone 10 to 600 s), wherein the insert provides the intensity variation of the 698 cm-1 along the SERS map. The main methotrexate intensity band profile significantly increases with the time of applying a constant potential, where the methotrexate band intensity reaches maximum intensity from the middle to the end of the map (Fig 2f inset). Furthermore, the increase in band intensity is also observed for the other methotrexate related bands at positions 1255 and 1461 cm-1.

Fig. 6 shows the EC-SERS band intensity at 698 cm-1 as a function of methotrexate concentration in an electrolyte solution comprising HCIO4 measured both with and without codeposition of Ag and halide ions, when applying a constant potential of 0.1 V for 420 s (so at time 600 s). The methotrexate detection is efficient already in a linear range between the concentration range between 0.25 to 2 pM. However, in the Ag and halide co-deposition experiment a clear performance improvement is observed. In addition, a higher reproducibility was observed for these measurements. In this way, in the Ag and halide codeposition measurements result in a detection range from 0.43-2.0 pM of methotrexate.

To further illustrate the methods and provide further characterization the provided substrates, a second round of experiments were performed. Specifically, this second round of experiments were found to further underline the above findings.

EXAMPLE 5

Characterisation of a SERS substrate comprising a gold surface portion on which a layer comprising a halide.

A first method for preparing a SERS substrate comprising a gold surface portion on which a layer comprising halide, said method comprises depositing, on the gold surface portion, by electrochemical deposition, a layer comprising silver halide. Fig. 7 schematically illustrates such first method for preparing a SERS substrate. More specifically, Fig. 7a shows the EC- SERS cell and sample chamber with electrodes and further illustrates the addition of electrolyte solution followed by electrochemical deposition of a layer comprising halide. Fig. 7b illustrates how the SERS substrate prepared in the EC-SERS sell can then be exposed to an analyte such as by drop casting followed by SERS mapping, wherein drop casting is used to refer to the method of placing a droplet comprising the analyte on the substrate followed by drying. Hence 7b. shows one way to use the provided substrate to obtain SERS mapping of a selected analyte.

To further characterise the layer comprising the halide, XPS analysis was performed on a substrate provided by the method for preparing a SERS substrate as of Fig. 7. Specifically, the SERS substrate was prepared by adding electrolyte solution of HCICU (500 pL [0.1 M]) without the analyte and applying five cycles of cyclic voltammetry using -0.6 V as the lowest potential, 1.3 V as the highest potential and a scan rate of 0.5 V/s to an EC-SERS cell comprises an AuNP substrate. Then, exchanging the electrolyte solution to an electrolyte solution comprising 0.1 M HCIO4, 0.5 pM Ag and 0.5 pM Br ions. Following this, two consecutive potentials were applied, more specifically a first potential of 0.9 V for 180 s and a second potential of 0.1 C for 420 s. The AuNP substrate comprises a layer comprising a halide was then dried and analysed with XPS (Fig 8-9).

XPS analysis was performed with Nexsa Surface Analysis System from Thermofisher Scientific. The data were acquired and further analysed with the software Avantage from Thermofisher Scientific. For the analysis, a monochromatic X-ray source of Al K-alpha photons at 1486.6 eV was used with a spot size of 400 pm. The high-resolution chemical characterization (Fig. 8) was performed with a pass energy of 50 eV, 10 scans, and a dwell time in each energy step of 50 ms. For the XPS survey (Fig 9) instead, a pass energy of 200 eV, 5 scans, and a dwell time in each energy step of 10 ms were used.

Fig. 8 shows the XPS peak analysis of selected atomic species measured on the AuNP substrate comprising a layer comprising a halide. Seen is further the XPS analysis of an electrochemically clean bare Au NP substrate here used as a reference. More specifically, Fig. 8a) shows the region of 360-380 eV comprising the Ag 3d peak region and Fig 8b) shows the region of 60-80 eV comprising the Br 3d peak region. For the AuNP substrate comprising a layer comprising a halide, Fig. 8a) shows the double peak of Ag 3d hereby confirming the presence of Ag on the substrate surface and similarly Fig. 8b) shows the double peak of Br 3d hereby confirming the presence of Br. The reference data of the clean bare AuNP substrate show no peaks in the Ag 3d or in the Br 3d regions (Fig 8).

Fig. 9 shows the XPS survey band analysis of the AuNP substrate comprising a layer comprising a halide. The band analysis shows peaks attributed to Au, Cu, Ag and Br, and further for C and O, hence again identifying the presence of Ag and Br on the substrate surface. Analysis of the XPS survey band further provides the atomic composition, hence atomic composition here shows Au 4f (44.4 %) to be the most abundant, and further provide the composition of Cu (13.5 %), Ag (less than 2 %), Br (2.9 %) and C Is (31.3 %), O Is (8.1%).

The provided substrate may further be used in a method for drug sensing, such as for detecting relevant therapeutic drug monitoring molecules i.e. analytes such as busulfan, imatinib and methotrexate, where an analyte to be tested is provided on the layer of the SERS substrate or in an electrolyte solution in contact with the layer of the SERS substrate and, wherein a SERS measurement is performed to sense the analyte. One way of doing this is illustrated in Fig. 7 b. Specifically, the AuNP substrate comprising a layer comprising a halide may be added an analyte using drop casting. Subsequently, SERS detection such as SERS mapping may be performed.

EXAMPLE 6

Repetition of a measurement of EC-SERS of busuifan on SERS substrate with Ag and halide ion co-deposition. Example 6 repeats a part of Example 2, however in Example 6 the solutions were prepared with less contamination, hence Example 6 provides cleaner datasets. Example 6 underlines the conclusions of Example 2, and thus confirms that busulfan detection below of 0.5 pM is enabled.

Fig. 10 shows a schematic illustration of the setup and method of this Example. More specifically, Fig. 7a shows the EC-SERS cell and sample chamber with electrodes and further illustrates the addition of electrolyte solution followed by electrochemical deposition of a layer comprising halide while in situ EC-SERS measurements are conducted. Fig. 7b shows an illustration of the substrate in an electrolyte solution comprising an analyte, halide ion and silver ions, said substrate, is illustrated before and after applying potential/ in situ EC-SERS measurements. After applying potential and in situ EC-SERS measurements, the AuNP substrate comprises a layer comprising a halide with an analyte.

Specifically, the repletion of the measurements of EC-SERS of busulfan on AuNP SERS substrate with and without Ag and halide ion co-deposition were obtained in the same way as of Example 2. The optimal analyte detection in acidic solution was performed in three steps: i) cleaning of the AuNP substrate by placing 500 pL of HCIO4 [0.1 M] without the analyte and applying five cycles of cyclic voltammetry using -0.6 V as the lowest potential, 1.3 V as the highest potential and a scan rate of 0.5 V/s; ii) preconditioning of the substrate, with the aim to reduce the hydrophobic property of the AuNP substrate, which was performed by replacing the HCIO4 cleaning solution with the HCIO4 electrolyte solution (500 pL [0.1 M]) containing the analyte, including or in the absence of Ag and Br ions, then the voltage is reduced to a first voltage of 0.9 V, in 0.2 V increment steps, to the 0.9 V which is applied for 180 s; iii) EC-SERS mapping was performed by the immediate change from 0.9 V to 0.1 V and simultaneous EC-SERS mapping along 420 s of continuously applied potential. The AgBr codeposition was simultaneously performed under the same EC-SERS parameters, by adding a solution of AgBr to reach a final concentration of Ag ions and Br ions of 0.5 pM. The concentration of the analyte, here busulfan, was between 0 and 2.0 pM. All measurements were performed in triplicates.

Fig. lla-c) and Fig 11 shows the data from the repetition measurement of an average SERS spectra of busulfan solutions at different concentrations using electrochemical co-deposition with Ag and halide ions (here bromide) on the AuNP substrate, when applying a constant potential of 0.1 V for 420 s. In Fig. 10 a) a clear and intense band at 1028 cm-1 is visible at 0.5 pM, and it becomes more intense with increasing busulfan concentrations. In addition, a band at 1028 cm-1 is visible at 0.1 pM busulfan. Consequently, this experiment confirms that busulfan detection below of 0.5 pM is enabled, even busulfan detection at 0.1 pM busulfan is enabled. Fig. 11 b) shows an EC-SERS map of busulfan band distribution (intensity as a function of wavenumber, where the wavenumber range specifically related to the highest intense band of the busulfan, the 1028 cm-1 band) along the applied potential from 180 to 600 s, using a busulfan concentration of 1.0 pM, and Ag and halide ion co-deposition. Fig. 10c) shows the intensity distribution as a function of wavenumber in the EC-SERS map along the applied potential time of 420 s (the time provided in time frames from 180 s to 600 s). The main busulfan intensity band profile increases with the time of applying a constant potential. The main busulfan band is observed intense from the beginning of the measurement - and continuously increases throughout the measurement. Furthermore, the increase in band intensity is also observed for the other busulfan related bands at 820, 859, 1176 and 1212 cm-1.

Fig. 12. shows calibration curves for Ag and halide ion (here bromide) co-deposition with busulfan in 0.1M HCIO4 under an applied potential of 0.1 V vs. Ag/ACI, band intensity at 1028 cm ¹ against different concentrations ranging from 0.1 pM to 5 pM. The curve is fitted by a sigmoidal curve (Hill equation) and the linear part of the calibration is shown in the inset with R2 correlation. Error bars represent the standard diviation (SD) of n=3 SERS substrate measurements. Specifically, the plot in the inset between 0.1 and 1 pM busulfan shows a linear correlation with a determination coefficient of correlation of R2 = 0.9912 with an average RSD of 20% over the linear range. In this way, when applying Ag and halide ion codeposition with busulfan, said method could detect busulfan below 0.5 pM such as of 0.1 pM. Additionally, the calibration curves shows that the concentration of busulfan may be determined down to as low as 0.5 pM. Consequently, the method provides detection of an analyte below the lower limit of the clinical suggested range (2-20 pM).

EXAMPLE 8

This example aims to characterise the SERS substrate after EC-SERS measurements of busulfan on AuNP SERS substrate with Ag and halide ion co-deposition i.e. characterised the produced substrate of Example 7.

XPS analysis was performed with Nexsa Surface Analysis System from Thermofisher Scientific. The data were acquired and further analysed with the software Avantage from Thermofisher Scientific (version 6.6). For the analysis, a monochromatic X-ray source of Al K- alpha photons at 1486.6 eV was used with a spot size of 400 pm. The high-resolution chemical characterization (Fig 13) was performed with a pass energy of 50 eV, 10 scans, and a dwell time in each energy step of 50 ms. For the XPS survey instead (Fig 14a), a pass energy of 200 eV, 5 scans, and a dwell time in each energy step of 10 ms were used. In addition, a composition depth profile analysis was performed on one Au NP substrate with Ag and halide ion (here bromide) electrochemical co-deposition (Fig. 14b). The depth profiling was performed with a monatomic ion source with Ar ion clusters of 4000 eV, with high current and an etch cycle time of 15 s for a total of 40 levels. For each level, the chemical characterization was performed in snapshot mode with a pass energy of 150 eV, 5 frames and 1 s per frame.

Fig. 13a-b) shows the XPS peak analysis of selected atomic species for the AuNP substrate after electrochemical Ag and Br co-deposition with the analyte, busulfan, and for the electrochemically clean bare Au NP substrate here used as reference. Fig. 13a) shows the double peak of Ag 3d hereby confirming the presence of Ag on the substrate surface and similarly Fig. 13b) shows the double peak of Br 3d hereby confirming the presence of Br.

Fig. 13c) shows a table providing the intensity obtained from the high-resolution chemical characterization peaks of Ag 3d, Br 3d and of Au 4f. From the intensity, the Au to Ag ratio is determined to be approx. 183 such meaning that Ag constitutes approx. 0.5% of the atomic composition relative to Au. In this way, the layer comprising the halide comprises less than 1.00% silver relative to the gold surface portion. The ratio of Au to Br is determined to be approx. 786 such meaning that Br constitutes approx. 0.12% of the atomic composition relative to Au. In this way, the layer comprising the halide comprises less than 0.25% halogen relative to the gold surface portion. Consequently, the layer comprising the halide comprises silver halide, the silver to halogen being in a ratio of between 1.5: 1 and 10: 1 such as 2: 1 to 6: 1 such as 3 : 1 to 5: 1 such as 4: 1.

Fig. 14a) shows the XPS survey spectrum of the AuNP substrate after electrochemical Ag and bromide co-deposition with busulfan. The spectrum shows peaks attributed to Au, Ag and Br, hence again confirms the presence of Ag and Br on the substrate surface. Fig. 14b) shows the etch level analysis of the 60-80 eV region comprising the Br 3d peak region. Only the outer most surface (i.e. at etch level 0) can a double peak attributed Br 3d be identified. Consequently, the layer comprising the halide can be characterised as a surface layer.

EXAMPLE 9

Further measurements at physiological pH Average SERS spectra for a busulfan concentration of 1 pM in a variety of electrolyte solutions also varying in pH, when applying a constant potential of 0.1 V. Specifically, SERS spectra were obtain of a substrate exposed to electrolyte solution comprising busulfan, a) wherein the electrolyte solution comprises phosphate-buffered saline (PBS) solution of pH 2.4, b) wherein the electrolyte solution comprises phosphate-buffered saline (PBS) solution of pH 7.4 and c) wherein the electrolyte solution comprises diluted ultrafiltered serum (commercial human serum) at human pH of 7.4. Specifically, the electrolyte solution comprising diluted ultrafiltered serum was prepared by ultra filtrating using a filter with a cutoff value of 10 kD. The SERS experiment was performed on substrate i) after electrochemical Ag and halide ion (here bromide) co-deposition with busulfan as well as on substrate ii) prepared with busulfan without Ag and halide ion co-deposition. As reference data also blank experiments were conducted, wherein busulfan was not present thus on substrates prepared in electrolyte solution with Ag and halide ions or for the ultrafiltered serum substrates prepared in ultrafiltered serum without Ag and halide ions.

Fig. 15a) shows SERS spectra of a substrate, wherein the electrolyte solution comprises phosphate-buffered saline (PBS) solution of pH 2.4. Fig. 15a) demonstrates that the band at 1028 cm-1 is still the most evident and, again, highly intense in the system containing Ag and halide ions (i.e. bromide) (data i)) relative to the system without Ag and halide ion (i.e. bromide) co-deposition (data ii).

Fig. 15b) shows SERS spectra of a substrate, wherein the electrolyte solution comprises phosphate-buffered saline (PBS) solution of pH 7.4. Fig. 15b) demonstrates that the band at 1028 cm-1 is most evident and, thus confirms the findings of Example 3, Fig 4. It is further found that the band at 1028 cm-1 is most distinct in the system containing Ag and halide ions (i.e. bromide) (data i)) relative to the system without Ag and halide ion (i.e. bromide) co-deposition (data ii).

Fig. 15c) shows SERS spectra of a substrate, wherein the electrolyte solution comprises ultra filtrated serum (commercial human serum) at human pH of pH 7.4. The band at 1028 cm-1 is clearly seen in the spectrum of the substrate in serum comprising busulfan with Ag and halide ion (i.e. bromide) co-deposition. The reference spectrum of the substrate in serum show no busulfan peak. In this way, the data shows a clear presence of busulfan and thus confirms that busulfan in a concentration of 1 pM is detectable. Consequently, the developed method can be applied under physiological simulated conditions, which allow for the applications of EC-SERS and Ag and halide co-deposition for sensitive and reproducible detection in serum under human pH value. EXAMPLE 10

Reusability of the EC-SERS substrate

EC-SERS mapping was performed on a AuNP substrate under Ag and halide ion co-deposition conditions, the busulfan concentration being of 1 pM in HCIO4. More specifically, AuNP substrate was provided in an electrolytic cell and 500 pL of HCIO4 without busulfan (i.e. cleaning solution) was applied. Five cycles of cyclic voltammetry were performed using the conditions of the high potential being 1.25 V, the low potential being -0.75 and the scan rate being 0.5 V/s in a range from -0.4 to 1.3 V. Specifically, the cycle was conducted by first increasing the potential from 0.0 V to 1.25 V, followed by decreasing the potential to -0.75 V, followed by increasing the potential to 0.1 V. The HCIO4 solution without busulfan was removed and replaced with the electrolyte solution comprising Ag and halide ions and further comprising busulfan in a concentration of 1 pM in HCIO4. Following this, the first preconditioning potential of 0.9 V was applied for 180 s followed by a switching to a second potential of 0.1 V, the second being applied for 420 s (for the co-deposition and the SERS measurement). The EC-SERS mapping was conducted immediately after initiating the second potential. Fig. 16a shows that the intensity of the main band at 1028 cm-1 is 1013 counts.

The electrolyte solution in the electrolytic cell was then exchanged to a cleaning electrolyte solution being a perchloric acid (HCIO4) solution and a cycle of cyclic voltammetry was preformed using the conditions of the high potential being 1.3 V, the low potential being -0.6 V and the scan rate being 0.5 V/s in a range from -0.4 to 1.3 V. Specifically, the cycle was conducted by first increasing the potential from 0.0 V to 1.3 V, followed by decreasing the potential to -0.6 V, followed by increasing the potential to 0.1 V. In this way, a first cleaning step (of the already used AuNP substrate) was performed. The cleaning electrolyte solution was then removed and SERS mapping of the Au substrate without applying any potential was performed. Fig. 16b shows that the intensity of the main band at 1028 cm-1 is 59 counts.

A second electrolyte solution was then applied to the gold surface portion of the AuNP substrate, the second electrolyte solution being a perchloric acid (HCIO4) solution comprising Ag/Br ions and 1 pM busulfan. The first preconditioning potential of 0.9 V was applied for 180 s followed by a switching to a second potential of 0.1 V, the second being applied for 420 s (for the co-deposition and the SERS measurement). The second EC-SERS mapping was conducted immediately after initiating the second potential. Fig. 16c shows that the intensity of the main band at 1028 cm-1 is 695 counts.

After acquiring the second EC-SERS mapping, a second cleaning step was performed using the same cleaning process as of the first cleaning step. Hence, the electrolyte solution in the electrolytic cell was exchanged to a cleaning electrolyte solution being a perchloric acid (HCIO4) solution (0.1 pM) and a cycle of cyclic voltammetry was preformed using the conditions of the high potential being 1.3 V, the low potential being -0.6 V and the scan rate being 0.5 V/s in a range from -0.4 to 1.3 V. Specifically, the cycle was conducted by first increasing the potential from 0.0 V to 1.3 V, followed by decreasing the potential to -0.6 V, followed by increasing the potential to 0.1 V. The cleaning electrolyte solution was then removed and SERS mapping of the Au substrate without applying any potential was performed. Fig. 16d shows that the intensity of the main band at 1028 cm-1 is 16 counts.

Fig. 16 shows the four SERS scans, wherein the intensity of the band at 1028 cm-1 changes for 1013 counts for the first EC-SERS measurement where the electrolyte comprises busulfan, and Ag/Br ions, to being reduced to 59 counts after the first cleaning step, and further where the intensity of the band at 1028 cm-1 changes from 695 counts for the second EC-SERS measurement where the electrolyte comprises busulfan, and Ag/Br ions, to being reduced to 16 counts after the second cleaning step. Consequently, it is seen that the method developed allow for reusability of the substrate as the cleaning step reduces the intensity of the main band to such an extent that the amount of analyte still detectable is below the intensity of the same band for a EC-SERS detection of busulfan of a 0.1 pM concentration (reference measurement may be found in Example 5, intensity of main band being approx. 100 counts). Hence reusing the substrate for a second measurement does not seem to significantly reduce the analyte detection levels.

Reusability of the substrate has the advantage that it allows for a series of measurements such as obtaining multiple datasets from a series of solutions, hereby removing uncertainties resulting from variation in the bare AuNP substrate.

In addition, this example may further provide evidence for that the layer comprising halide comprises such as essentially consist of a nanometric ionic layer. Applying a cleaning step (i.e. a cycle of cyclic voltammetry) results in busulfan leaving the SERS substrate as busulfan signal is significantly decreased. As busulfan seems to interact primarily with silver ions, silver ions may further be leaving together with busulfan. Had the layer comprising halide been metallic we would not have expected the substrate to reach a state of binding such low amounts of busulfan as detected.

EXAMPLE 11

This experiment serves as a comparative example indicating a poor detection limit when busulfan is added to an AgNP substrate using drop casting. A series of electrolyte solutions comprising busulfan in a range of concentrations was prepared. More specifically, the electrolyte solutions were prepared in phosphate-buffered saline (PBS) pH 7.4 with the busulfan ranging from 0.5 pM-20 pM.

Fig. 17 a) shows two EC-SERS maps of the main busulfan band, the first map in the top showing a freshly dropped busulfan [20 pM] solution on the Ag NP substrate, while it was drying and the second map in the bottom showing the same substrate after it was thoroughly dried. The arrow indicates the drying step along the length of the entire mapping, the duration of the mapping being 3 min. The thoroughly dried substrate shows a drop pattern and an approx., four times reduction in band intensity. This drop in intensity may be attributed to a rearrangement of the busulfan at the surface, possibly coursed by replacement with ions from the electrolyte solution (i.e. phosphate ions). A drying effect is also seen over the 3 min in the top. Consequently, this method may result in significant uncertainty on the determined concentration levels of busulfan in the electrolyte solution as the evaluated concentration level might depend on the drying state of the sample when measured.

Fig. 17 a) further shows the busulfan band variations of the averaged spectrum at different concentrations, the inset highlighting the main busulfan band at 1028 cm^-1. The band intensity variations reflect the decrease in intensity while reducing the busulfan concentration. The data indicates that the busulfan can be detected using this method when the busulfan concentration is of 1 pM where as a busulfan concentration of 0.5 pM does not provide clear busulfan detection.

Fig. 17 b) shows a calibration curve built on the main busulfan band intensity at different concentrations (Fig. 17a). Specifically, the data shows the average spectrum of 3 measurements and error bars representing the standard deviation, n=3 ±SD. The calibration curve shows a linear correlation between busulfan concentration and band intensity from 2.5 to 20 pM. However, the average relative standard deviation (RDS) is above 40 showing a high degree of variation in sensitivity, hence indicating a limited degree of reproducibility of the measurements. This limited degree of reproducibility is a result of first, the variation in time between placing the drop on the surface and the SERs map acquisition between measurements and secondly, the variation in room temperature and humidity during measurements.

Consequently, this example shows a detection limit of above 0.5 pM busulfan or of approx. 0.5 pM for busulfan, when busulfan is added to an AgNP substrate using drop casting. The method of drop casting is further shown to provide large variation in sensitivity, hence a limited degree of reproducibility. ASPECTS

1. A SERS substrate comprising a gold surface portion on which a layer comprising a halide is provided.

2. The SERS substrate according to aspect 1, wherein the layer comprising the halide is a monolayer amount of halide.

3. The SERS substrate according to any one of aspects 1 or 2, wherein the gold surface portion defines a plurality of nanopillars.

4. A SERS substrate according to any one of the preceding aspects, wherein the layer comprising the halide comprises a silver halide.

5. The SERS substrate according to aspect 4, wherein the layer comprising the silver halide is a monolayer amount of silver halide.

6. The SERS substrate according any one of aspects 4 or 5, wherein the layer comprising silver halide comprises silver chloride (AgCI), silver bromide (AgBr), and/or silver iodide (Agl), preferably silver bromide (AgBr).

7. A method for the manufacture of a SERS substrate, the method comprising : providing a SERS substrate with a gold surface portion, applying, to the gold surface portion, an electrolyte solution comprising a halide ion source and depositing on the gold surface portion, by electrochemical deposition, an ionic layer comprising halide.

8. The method according to aspect 7, wherein the depositing step comprises, subsequent to providing the electrolyte solution, providing a voltage to the SERS substrate, between the SERS substrate as a working electrode, and a reference electrode.

9. The method according to aspect 8, wherein the reference electrode is an Ag/AgCI reference electrode.

10. The method according to aspect 7 or 8, wherein the voltage is above a threshold voltage during a deposition time interval of 180-800 s.

11. The method according to any of aspects 8 to 10, where the voltage applied is in the range of 1.3 V to 0.4 V. 12. The method according to any one of aspect 7 to 9, wherein the electrolyte solution additionally comprises a silver ion source and wherein the deposition step comprises depositing, on the gold surface portion, by electrochemical deposition, an ionic layer comprising a silver halide.

13. The method according to aspect 12, wherein the method further comprises: a. subsequent to applying, to the gold surface portion, the electrolyte solution; providing a first voltage to the SERS substrate, wherein said first voltage is either above or below a threshold voltage. b. subsequent to step a. providing a second voltage to the SERS substrate, wherein if said first voltage is above the threshold voltage, said second voltage is below the threshold voltage or if said first voltage is below the threshold voltage said second voltage is above the threshold voltage.

14. The method according to aspect 13, wherein the reference electrode is an Ag/AgCI reference electrode, the first voltage is in the interval 0.4 to 1.3 V and is applied in a deposition time interval of 160-200 s, and the second voltage is in the interval of -0.8 to 0.2 V and is applied in a deposition time interval of 180-800 s.

15. The method according to aspect 13, wherein: the reference electrode is an Ag/AgCI reference electrode, the first voltage is in the interval -0.8 to 0.2 V and is applied in a deposition time interval of 160-200 s, and the second voltage is in the interval 0.4 to 1.3 V of and is applied in a deposition time interval of 180-800 s.

16. The method of any one of aspect 12-15, wherein the silver halide comprises silver chloride (AgCI), silver bromide (AgBr), and/or silver iodide (Agl), preferably silver bromide (AgBr).

17. The method of any one of aspect 12-16, wherein the silver halide comprises chlorine, bromine, iodine, preferably bromine. 18. The method according to any one of aspects 7-17, wherein the electrolyte solution comprises a concentration of halide ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM.

19. The method according to any one of aspects 12-18, wherein the electrolyte solution comprises a concentration of silver ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM.

20. The method according to any one of aspects 7-19, wherein said electrolyte solution is an acidic solution, such as a perchloric acid (HCIO4) solution, a sulfuric acid solution, or a hydrochloric acid solution; or a buffered solution, such as a phosphate-buffered saline (PBS) solution; or a human serum solution such as an ultrafiltered human serum.

21. The method according to any one of aspects 7-20, wherein said electrolyte solution is a buffered solution, such as a phosphate-buffered saline (PBS) solution; or a human serum solution such as an ultrafiltered human serum having a pH of between 7.0-8.0, such as 7.0- 7.8, such as of 7.2-7.6, such as 7.4.

22. The method according to any one of aspect 7-21, wherein the gold surface portion defines a plurality of nanopillars.

23. A method of performing a SERS measurement for detection or sensing of an analyte, wherein said method comprises: providing a SERS substrate with a gold surface portion, applying, to the gold surface portion, an electrolyte solution comprising a halide ion source and the analyte and simultaneously depositing on the gold surface portion, by electrochemical deposition, an ionic layer comprising halide and performing the SERS measurement.

24. The method of performing a SERS measurement according to aspect 23, wherein the method further comprises an electrolyte solution comprising a silver ion source, wherein the method further comprises simultaneously depositing on the gold surface portion, by electrochemical deposition, an ionic layer comprising silver halide and performing the SERS measurement.

25. The method according to any one of aspect 23-24, wherein the method further comprises: a. subsequent to applying, to the gold surface portion, the electrolyte solution; providing a first voltage to the SERS substrate, wherein said first voltage is either above or below a threshold voltage. b. subsequent to step a. providing a second voltage to the SERS substrate, wherein if said first voltage is above the threshold voltage, said second voltage is below the threshold voltage or if said first voltage is below the threshold voltage said second voltage is above the threshold voltage.

26. The method according to aspect 25, wherein the reference electrode is an Ag/AgCI reference electrode, the first voltage is in the interval 0.4 to 1.3 V and is applied in a deposition time interval of 160-200 s, and the second voltage is in the interval of -0.8 to 0.2 V and is applied in a deposition time interval of 180-800 s.

27. The method according to aspect 25, wherein: the reference electrode is an Ag/AgCI reference electrode, the first voltage is in the interval -0.8 to 0.2 V and is applied in a deposition time interval of 160-200 s, and the second voltage is in the interval 0.4 to 1.3 V of and is applied in a deposition time interval of 180-800 s.

28. The method according to any one of aspect 23-27, wherein the layer comprising halide comprises chlorine, bromine, iodine, preferably bromine.

29. The method according to any one of aspect 23-28, wherein the layer comprising silver halide comprises silver chloride (AgCI), silver bromide (AgBr), and/or silver iodide (Agl), preferably silver bromide (AgBr).

30. The method according to any one of aspects 23-29, wherein the electrolyte solution comprises a concentration of halide ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM.

31. The method according to any one of aspects 23-30, wherein the electrolyte solution comprises a concentration of silver ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM. 32. The method according to any one of aspects 23-31, wherein said electrolyte solution is an acidic solution, such as a perchloric acid (HCIO4) solution, a sulfuric acid solution, or a hydrochloric acid solution; or a buffered solution, such as a phosphate-buffered saline (PBS) solution; or a human serum solution such as an ultrafiltered human serum.

33. The method according to any one of aspect 23-32, wherein the gold surface portion defines a plurality of nanopillars.

34. The method according to any one of aspect 23-32, wherein the method further comprises performing a second SERS measurement for detection or sensing of an second analyte, wherein said method comprises the SERS substrate with a gold surface portion after a SERS measurement, applying, to the gold surface portion, at least one cycle of cyclic voltammetry, further applying, to the gold surface portion, a second electrolyte solution comprising a silver ion source, a halide ion source and the second analyte and simultaneously depositing on the gold surface portion, by electrochemical deposition, a layer comprising halide and performing the second SERS measurement.

35. The method according to aspect 34, wherein the method further comprises exchanging the electrolyte solution applied, to the gold surface portion, to a cleaning electrolyte solution prior to the at least one cycle of cyclic voltammetry.

36. The method according to any one of aspect 34-35, wherein said cleaning electrolyte solution consists of an acidic solution, such as a perchloric acid (HCIO4) solution, a sulfuric acid solution, or a hydrochloric acid solution; or a buffered solution, such as a phosphate- buffered saline (PBS) solution.

37. The method according to any one of aspect 34-36, wherein the second electrolyte solution further comprises a concentration of silver ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM and/or a concentration of halide ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM.

38. The method according to any one of aspect 34-37, wherein the second electrolyte solution is an acidic solution, such as a perchloric acid (HCIO4) solution, a sulfuric acid solution, or a hydrochloric acid solution; or a buffered solution, such as a phosphate-buffered saline (PBS) solution; or a human serum solution such as an ultrafiltered human serum.

39. A SERS substrate manufacturing kit, comprising a SERS substrate with a gold surface portion, a halide ion source and optionally a silver ion source. 40. The SERS substrate manufacturing kit according to aspect 39, wherein gold surface portion defines a plurality of nanopillars.

41. The SERS substrate manufacturing kit according to any one of aspect 39 or 40, wherein the silver ion source and the halide ion source comprise silver chloride (AgCI), silver bromide (AgBr), and/or silver iodide (Agl), preferably silver bromide (AgBr).

42. The SERS substrate manufacturing kit according to any one of aspect 39 to 41, said kit further comprising an electrolyte solution, said electrolyte solutions being an acidic solution, such as a perchloric acid (HCIO4) solution, a sulfuric acid solution, or a hydrochloric acid solution; or a buffered solution, such as a phosphate-buffered saline (PBS) solution; or a human serum solution such as an ultrafiltered human serum.

43. The SERS substrate manufacturing kit according to any one of aspect 39 to 42, wherein said electrolyte solution comprises the electrolyte, the electrolyte being perchloric acid, sulfuric acid, hydrochloric acid, phosphate in a concentration of 0.05-0.15M such as 0.1 M.

44. The SERS substrate manufacturing kit according to any one of aspect 39 to 43, wherein the electrolyte solution comprises a concentration of halide ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM.

45. The SERS substrate manufacturing kit according to any one of aspect 39 to 44, wherein the electrolyte solution comprises a concentration of silver ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM.

46. Use of a SERS substrate manufacturing kit as provided by any one of aspect 39-45, wherein said kit may be used to obtain a SERS substrate with a gold surface on which an electrochemically deposited layer comprising silver halide is provided.

47. A SERS substrate comprising a gold surface portion on which a layer comprising a halide is provided, wherein the layer comprising the halide is less than 2.0 nm.

48. The SERS substrate according to aspect 47, wherein the gold surface portion defines a plurality of nanopillars.

49. The SERS substrate according to any one of aspect 47-48, wherein the layer comprising the halide is a nanometric ionic layer. 50. The SERS substrate according to any one of aspect 47-49, wherein the layer comprising the halide comprises less than 0.25% halogen relative to the gold surface portion.

51. The SERS substrate according to any one of aspect 47-50, wherein the surface layer comprising the halide comprises chlorine, bromine, iodine, preferably bromine.

52. The SERS substrate according to any one of aspect 47-51, wherein the layer comprising the halide comprises silver.

53. The SERS substrate according to any one of aspect 47-52, wherein the layer comprising the halide comprises less than 1.00% silver relative to the gold surface portion.

54. The SERS substrate according to any one of aspect 47-53, wherein the layer comprising the halide comprises silver halide, the silver to halogen being in a ratio of between 1.5: 1 and 10: 1 such as 2: 1 to 6: 1 such as 3: 1 to 5: 1 such as 4: 1.

55. Use of a SERS substrate according to any one of aspects 1-6 or any one of aspect 47- 54 in a method for drug sensing, such as for detecting relevant therapeutic drug monitoring molecules i.e. analytes such as imatinib, busulfan and methotrexate, where an analyte to be tested is provided on the layer of the SERS substrate or in an electrolyte solution in contact with the layer of the SERS substrate and, wherein a SERS measurement is performed to sense the analyte.

Claims

1. A method for the manufacture of a SERS substrate, the method comprising : providing a SERS substrate with a gold surface portion, applying, to the gold surface portion, an electrolyte solution comprising a halide ion source and depositing on the gold surface portion, by electrochemical deposition, a layer comprising halide.

2. The method according to claim 1, wherein the depositing step comprises, subsequent to providing the electrolyte solution, providing a voltage to the SERS substrate, between the SERS substrate as a working electrode, and a reference electrode.

3. The method according to claim 2, wherein: the reference electrode is an Ag/AgCI reference electrode, the voltage is above a threshold voltage during a deposition time interval of 180-800 s, the voltage applied is in the range of 1.3 V to 0.4 V, the gold surface portion defines a plurality of nanopillars, and/or the electrolyte solution additionally comprises a silver ion source and wherein the deposition step comprises depositing, on the gold surface portion, by electrochemical deposition, a layer comprising silver halide.

4. The method according to claim 2, wherein the electrolyte solution additionally comprises a silver ion source and wherein the deposition step comprises depositing, on the gold surface portion, by electrochemical deposition, a layer comprising silver halide, wherein the method further comprises: a. subsequent to applying, to the gold surface portion, the electrolyte solution; providing a first voltage to the SERS substrate, wherein said first voltage is either above or below a threshold voltage. b. subsequent to step a. providing a second voltage to the SERS substrate, wherein if said first voltage is above the threshold voltage, said second voltage is below the threshold voltage or if said first voltage is below the threshold voltage said second voltage is above the threshold voltage.

5. The method according to claim 4, wherein the layer comprising silver halide, said silver halide comprises halogen selected from chlorine, bromine and/or iodine, preferably wherein the halogen is bromine, and/or wherein the gold surface portion defines a plurality of nanopillars and/or wherein the layer comprising silver halide further comprises copper.

6. A method of performing a SERS measurement for detection or sensing of an analyte, wherein said method comprises: providing a SERS substrate with a gold surface portion, applying, to the gold surface portion, an electrolyte solution comprising the analyte and a halide ion source and simultaneously depositing on the gold surface portion, by electrochemical deposition, a layer comprising halide and performing the SERS measurement.

7. The method according to claim 6, wherein the method further comprises an electrolyte solution comprising a silver ion source, wherein the method further comprises simultaneously depositing on the gold surface portion, by electrochemical deposition, a layer comprising silver halide and performing the SERS measurement.

8. The method according to any one of claims 6-7, wherein the method further comprises an electrolyte solution comprising a copper ion source, wherein the method further comprises simultaneously depositing on the gold surface portion, by electrochemical deposition, a layer comprising copper halide and performing the SERS measurement.

9. The method according to any one of claim 6-8, wherein the method further comprises: a. subsequent to applying, to the gold surface portion, the electrolyte solution; providing a first voltage to the SERS substrate, wherein said first voltage is either above or below a threshold voltage. b. subsequent to step a. providing a second voltage to the SERS substrate, wherein if said first voltage is above the threshold voltage, said second voltage is below the threshold voltage or if said first voltage is below the threshold voltage said second voltage is above the threshold voltage.

10. The method according to claim 9, wherein the reference electrode is an Ag/AgCI reference electrode, the first voltage is in the interval 0.4 to 1.3 V and is applied in a deposition time interval of 160-200 s, and the second voltage is in the interval of -0.8 to 0.2 V and is applied in a deposition time interval of 180-800 s.

11. The method according to claim 9, wherein: the reference electrode is an Ag/AgCI reference electrode, the first voltage is in the interval -0.8 to 0.2 V and is applied in a deposition time interval of 160-200 s, and the second voltage is in the interval 0.4 to 1.3 V of and is applied in a deposition time interval of 180-800 s.

12. The method according to any one of claims 6-11, wherein the layer comprising halide comprises chlorine, bromine, iodine, preferably bromine.

13. The method according to any one of claims 6-12, wherein the layer comprising silver halide comprises silver chloride (AgCI), silver bromide (AgBr), and/or silver iodide (Agl), preferably silver bromide (AgBr).

14. The method according to any one of claims 6-13, wherein the electrolyte solution comprises a concentration of halide ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM.

15. The method according to any one of claims 6-14, wherein the electrolyte solution comprises a concentration of silver ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM.

16. The method according to any one of claims 6-15, wherein said electrolyte solution is an acidic solution, such as a perchloric acid (HCIO4) solution, a sulfuric acid solution, or a hydrochloric acid solution; or a buffered solution, such as a phosphate-buffered saline (PBS) solution; or a human serum solution such as an ultrafiltered human serum.

17. The method according to any one of claims 6-16, wherein the gold surface portion defines a plurality of nanopillars.

18. The method according to any one of claims 6-17, wherein the method further comprises performing a second SERS measurement for detection or sensing of a second analyte, wherein said method comprises the SERS substrate with a gold surface portion after a SERS measurement, applying, to the gold surface portion, at least one cycle of cyclic voltammetry, further applying, to the gold surface portion, a second electrolyte solution comprising a silver ion source, a halide ion source and the second analyte and simultaneously depositing on the gold surface portion, by electrochemical deposition, a layer comprising halide and performing the second SERS measurement.

19. The method according to claim 18, wherein the method further comprises exchanging the electrolyte solution applied, to the gold surface portion, to a cleaning electrolyte solution prior to the at least one cycle of cyclic voltammetry.

20. The method according to any one of claims 18-19, wherein said cleaning electrolyte solution consists of an acidic solution, such as a perchloric acid (HCIO4) solution, a sulfuric acid solution, or a hydrochloric acid solution; or a buffered solution, such as a phosphate- buffered saline (PBS) solution.

21. The method according to any one of claims 18-20, wherein the second electrolyte solution further comprises a concentration of silver ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM and/or a concentration of halide ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM.

22. The method according to any one of claims 18-21, wherein the second electrolyte solution is an acidic solution, such as a perchloric acid (HCIO4) solution, a sulfuric acid solution, or a hydrochloric acid solution; or a buffered solution, such as a phosphate-buffered saline (PBS) solution; or a human serum solution such as an ultrafiltered human serum.

23. A SERS substrate manufacturing kit, comprising a SERS substrate with a gold surface portion, a halide ion source and optionally a silver ion source.

24. The SERS substrate manufacturing kit according to claim 23, wherein gold surface portion defines a plurality of nanopillars, and/or wherein the silver ion source and the halide ion source comprise silver chloride (AgCI), silver bromide (AgBr), and/or silver iodide (Agl), preferably silver bromide (AgBr).

25. The SERS substrate manufacturing kit according to any one of claims 23-24, said kit further comprising an electrolyte solution, said electrolyte solutions being an acidic solution, such as a perchloric acid (HCIO4) solution, a sulfuric acid solution, or a hydrochloric acid solution; or a buffered solution, such as a phosphate-buffered saline (PBS) solution; or a human serum solution such as an ultrafiltered human serum.

26. The SERS substrate manufacturing kit according to any one of claims 23-25, wherein said electrolyte solution comprises the electrolyte, the electrolyte being perchloric acid, sulfuric acid, hydrochloric acid, phosphate in a concentration of 0.05-0.15M such as 0.1M.

27. The SERS substrate manufacturing kit according to any one of claims 23-26, wherein the electrolyte solution comprises a concentration of halide ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM.

28. The SERS substrate manufacturing kit according to any one of claims 23-27, wherein the electrolyte solution comprises a concentration of silver ions equal to or below 1.0 pM, such as between 1.0 to 0.1 pM, preferably of around 0.5 pM.

29. Use of a SERS substrate manufacturing kit as provided by any one of claims 23-28, wherein said kit may be used to obtain a SERS substrate with a gold surface on which an electrochemically deposited layer comprising silver halide is provided.

30. A SERS substrate comprising a gold surface portion on which a layer comprising halide is provided.

31. The SERS substrate according to claim 30, wherein the gold surface portion defines a plurality of nanopillars.

32. The SERS substrate according to any one of claims 30-31, wherein the layer comprising halide is a nanometric ionic layer.

33. The SERS substrate according to any one of claims 30-32, wherein the layer comprising halide has a thickness of less than 2.0 nm.

34. The SERS substrate according to any one of claims 30-33, wherein the layer comprising halide comprises less than 0.25% halogen relative to the gold surface portion.

35. The SERS substrate according to any one of claims 30-34, wherein the layer comprising halide comprises chlorine, bromine, iodine, preferably bromine.

36. The SERS substrate according to any one of claims 30-35, wherein the layer comprising halide comprises silver.

37. The SERS substrate according to any any one of claims 30-36, wherein the layer comprising halide comprises less than 1.00% silver relative to the gold surface portion.

38. The SERS substrate according to any one of claims 30-37, wherein the layer comprising halide comprises silver halide, the silver to halogen being in a ratio of between 1.5: 1 and 10: 1 such as 2: 1 to 6:1 such as 3: 1 to 5: 1 such as 4:1.

39. Use of a SERS substrate according to any one of claims 30-38 in a method for drug sensing, such as for detecting relevant therapeutic drug monitoring molecules i.e. analytes such as imatinib, busulfan and methotrexate, where an analyte to be tested is provided on the layer of the SERS substrate or in an electrolyte solution in contact with the layer of the SERS substrate and, wherein a SERS measurement is performed to sense the analyte.