SERS method for ergothioneine quantification by surface-amplified raman scattering spectroscopy

Abstract

To provide a reliable, rapid, sensitive, and efficient analytical method for quantitative determination of EG in various matrices, more specifically, a method that enables quantitative measurement of EG in any type of samples in a precise and accurate manner.SOLUTION: The present invention relates to a method of detecting and / or determining the amount of ergothioneine (EG) in a sample using surface-enhanced Raman scattering (SERS) spectroscopy. The method involves the use of an internal standard represented by a formula (I) or (II) as explained in the description herein. The internal standard may preferably be 5-amino-2-mercaptobenzimidazole (5A2MBI).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the field of systems and methods for detecting the presence and / or determining the quantitative amount of biomolecules using Surface Enhanced Raman Scattering (SERS) spectroscopy, and in particular to a SERS method suitable for the quantitative determination of ergothioneine (EG) in a sample. [Background technology]

[0002] Ergothioneine is a dietary amino acid that has attracted much attention due to its excellent antioxidant properties and the potential health benefits it provides [1]. Ergothioneine stands out as a highly soluble and stable compound, and it exhibits exceptional resistance to heat and oxygen. Protecting cells from photochemical damage, neutralizing harmful reactive oxygen and nitrogen species (ROS and RNS), and reducing associated oxidative stress are all important biological functions of this compound [2]. Furthermore, ergothioneine has been linked to various physiological functions in humans, such as neuroprotection, anti-inflammatory, and anti-aging effects. As a result, ergothioneine has gained popularity as a promising ingredient in various applications, such as antioxidant formulations, skin care and cosmetics, dietary supplements, functional foods and beverages, pharmaceuticals, biotechnology, bioengineering, and clinical diagnostics [3,4]. The uses of ergothioneine as an ingredient continue to expand as research uncovers its wide range of benefits and applications. Its versatility, stability and strong antioxidant properties make it an attractive option for a wide range of industries looking to develop products with improved functionality, health benefits and consumer appeal.

[0003] Considering these diverse potential applications, there is an increasing demand for specific, simple, and easy methods to measure ergothioneine in various complex matrices such as food, cosmetics, and biological samples. Furthermore, accurate quantification of ergothioneine levels in biological samples is crucial for understanding its role in diseases associated with oxidative stress, such as neurodegenerative disorders, cardiovascular diseases, and cancer [1].

[0004] Existing analytical methods for ergothioneine determination include i) capillary electrophoresis [5], ii) high performance liquid chromatography (HPLC) [5,6], iii) inductively coupled plasma tandem mass spectrometry (HPLC-ICP-QQQ-MS) [7], and iv) liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MS / MS) [8]. However, all these techniques require complex and destructive sample preparation procedures, time-consuming separations, and high purity reagents, but lack the sensitivity required to detect low concentrations of ergothioneine in biological fluids. There is a need for reliable, rapid, sensitive, and efficient analytical methods that allow quantitative determination of ergothioneine in various sample matrices.

[0005] One approach that has been effectively used to measure compounds at concentrations similar to those present in biological fluids by EGs uses surface-enhanced Raman scattering (SERS). SERS spectroscopy has emerged as a powerful analytical technique for the detection and quantification of a variety of molecules [9]. SERS exploits the phenomenon of enhanced Raman scattering signal when an analyte adsorbs to nanostructured metal surfaces, such as gold or silver nanoparticles. The signal enhancement occurs due to the excitation of localized surface plasmon resonance on the metal nanoparticles, causing an enhancement of the Raman signal. However, one of the major challenges in accurate quantification using SERS is the variability that can occur during sample preparation and analysis

[10] . Factors such as variations in the SERS substrate, laser power, and experimental conditions can affect the Raman signal intensity, resulting in inaccurate quantification. Including an internal standard (IS) of known concentration in an unknown sample and using its SERS spectral band intensities as a reference for the band intensities of chemicals at unknown concentrations is an effective way to limit such variability errors. However, the variability in signal enhancement among various analytes makes it difficult to identify a single compound that can serve as a suitable IS in every case. So far, no IS specific for ergothioneine has been reported in the literature.

[0006] Therefore, there remains a need for reliable, rapid, sensitive and efficient analytical methods for the quantitative determination of EG in various matrices, more specifically, a method capable of measuring the amount of EG in any kind of sample in a precise and accurate manner is required. Summary of the Invention

[0007] To address the technical problems associated with the previously mentioned methods, the inventors of the present invention provide an effective and reliable method for the rapid detection and measurement of ergothioneine (EG) using surface-enhanced Raman scattering (SERS) spectroscopy and the addition of a specific internal standard for ergothioneine. Advantageously, the method involves the addition of a compound of formula (I), such as 5-amino-2-mercaptobenzimidazole (5A2MBI), as an internal standard for ergothioneine, which helps to reduce the variation in SERS signal arising from different SERS substrates.

[0008] The object of the present invention is to provide a method for producing a method for the treatment of a disease comprising the steps of: a) adding at least one internal standard (IS) to a sample to obtain a sample containing at least one IS; b) adding at least one SERS substrate to the sample containing IS obtained in a); c) exposing the sample together with the SERS substrate obtained in step b) to a laser to generate a SERS spectrum; 1. A method for detecting and / or determining the amount of ergothioneine (EG) in a sample using surface-enhanced Raman spectroscopy (SERS), comprising: The internal standard has the following formula (I):

[0009] [ka]

[0010] (Wherein, R1 and R2 are each independently C 1~3 -Alkyl, NH2, NHCOC 1~3 Alkyl, NH(C 1~3-alkyl), N(C 1~3 -alkyl), halogen, aryl, and heteroaryl; or R and R are linked together and each is optionally selected from C 1~3 -Alkyl, NH2, NHCOC 1~3 Alkyl, NH(C 1~3 -alkyl), N(C 1~3 -alkyl), halogen, NO, CN; R3 is H, CH3, or NH2.

[0011] In one embodiment, the internal standard has formula (I), wherein R1 and R2 are joined to form a fused aromatic ring, in particular a phenyl optionally substituted with one or more substituents, preferably substituted with NH2, and / or R3 is H.

[0012] In a preferred embodiment, the internal standard has the following formula (II):

[0013] [ka]

[0014] (Wherein, R3 is H, CH3, or NH2; R4 is NH2, NO2, H, or a halogen.

[0015] Preferably, R3 is H.

[0016] Preferably, R4 is NH2.

[0017] In an even more preferred embodiment, the internal standard is 5-amino-2-mercaptobenzimidazole (5A2MBI). According to one aspect of the invention, in step a), the internal standard is added at a concentration comprised between 2.5 nM and 25 μM, preferably at a concentration of 0.25 μM.

[0018] According to one embodiment of the invention, in step c) a laser is used with a wavelength comprised between 600 nm and 850 nm, preferably 785 nm.

[0019] According to one aspect of the present invention, the method comprises determining the presence and / or amount of EG in a sample by measuring the concentration of EG at 484 cm -1 The additional step d) is to determine the characteristic spectral band of EG at 391-393 cm by comparing the characteristic spectral band of the internal standard. For 5A2MBI, the characteristic spectral band is 391-393 cm -1 It is.

[0020] According to one embodiment of the present invention, the SERS substrate comprises at least one nanostructure selected from spheroidal nanoparticles, nanorods, nanostars, and nanoplates.

[0021] According to one embodiment of the invention, the SERS substrate comprises at least one nanostructure of silver or gold reduced with citric acid or another reducing agent, or at least one nanostructure of silver or gold obtained by laser ablation.

[0022] In a preferred embodiment, the SERS substrate comprises citrate-reduced silver nanoparticles (c-AgNPs).

[0023] According to one aspect of the invention, the sample is selected from an isolated biological sample, such as a body fluid, a foodstuff, or a cosmetic product.

[0024] In a preferred embodiment, the sample is a body fluid, preferably serum.

[0025] According to one embodiment of the invention, the method also comprises a step of deproteinization of the sample prior to step a).

[0026] According to one embodiment of the invention, after step b), the sample together with the SERS substrate is placed, preferably in the form of one or more drops, on a support and left to dry until dry.

[0027] According to one embodiment of the invention, said support is selected from calcium fluoride plates and glass plates, preferably coated with at least one layer of aluminum or at least one layer of aluminum and at least one layer of parafilm.

[0028] Another object of the present invention is the use of a compound of formula (I) or (II) as defined above, preferably 5A2MBI, as an internal standard in a SERS method for determining the concentration of EG in a sample. [Brief description of the drawings]

[0029] [Figure 1] FIG. [Diagram 2] Figure 1 shows the dependence of the SERS signal on the concentration of EG in human serum. Each concentration level (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μM) was repeated five times over two days. The concentration of IS was 0.25 μM. [Diagram 3] Calibration curve for quantification of EG in human serum. Confidence bands are shown as dark grey and prediction bands as light grey shaded areas. The limit of detection (LOD) and limit of quantification (LOQ) are listed at the top left. The coefficient of determination (R2) value is 0.98. [Figure 4] FIG. [Diagram 5] Figure 1 shows the dependence of the SERS signal on the concentration of EG in human serum. Each concentration level (0, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2 μM) was replicated five times over two days. The concentration of IS was 0.25 μM. [Figure 6] Calibration curve for quantification of EG in human serum. Confidence bands are shown as dark grey and prediction bands as light grey shaded areas. Limits of detection (LOD) and limits of quantification (LOQ) are listed at the top left. The coefficient of determination (R2) value is 0.97. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] definition In the present invention, "ergothioneine" refers to the amino acid known by the IUPAC name (2S)-3-(2-sulfanylidene-2,3-dihydro-1H-imidazol-4-yl)-2-(trimethylazaniumyl)propanoate, also referred to herein by the acronyms "EG" or "EGT".

[0031] For the purposes of this invention, "surface-enhanced Raman scattering" (SERS) refers to an analytical method for the detection and quantification of compounds of interest in a sample that exploits the phenomenon of Raman scattering enhancement when the analyte is adsorbed onto a nanostructured metal surface. "SERS" is used synonymously herein.

[0032] In the present invention, "internal standard" means a compound that is added in a known amount to samples, blanks, and calibration standards in a chemical analysis.

[0033] In the present invention, "SERS substrate" means a substrate suitable for use in a SERS method, comprising a nanostructured surface of at least one noble metal. The metal can be selected from gold, silver, copper, or a combination thereof. In particular, the SERS substrate may comprise a multiplicity of nanostructures, a nanostructure being defined as a particle having a diameter or at least one length typically comprised between 1 and 100 nm, for example 50 nm. The nanostructures may have different shapes, in particular spherical, rod-shaped (nanorods) or star-shaped (nanostars). The nanostructures may be in aqueous dispersion or may be placed on a support, which may comprise paper, silicon, or other suitable material. In a preferred embodiment, the nanostructures are nanoparticles.

[0034] For the purposes of the present invention, "limit of detection (LOD)" refers to the lowest concentration of EG in a sample that can be consistently detected with a stated confidence level (typically 95%).

[0035] In the present invention, "limit of quantification (LOQ)" means the lowest concentration of EG in a sample that can be quantitatively detected with acceptable accuracy and precision (10% error).

[0036] For the purposes of this invention, "laser" means an optoelectronic device capable of emitting a coherent beam of light.

[0037] In the present invention, an "isolated biological sample" means a sample previously isolated from a subject. In particular, said sample may be, for example, a body fluid or a tissue.

[0038] For the purposes of the present invention, "body fluid" or "biological fluid" refers to any bodily fluid, such as blood, plasma, serum, saliva, or urine.

[0039] The method of the present invention has been validated on samples containing different concentrations of EG and achieved narrow confidence and prediction bands, indicating low model error.

[0040] In a preferred embodiment, the SERS substrate is a nanostructure selected from spherical nanoparticles, nanorods, nanostars, or nanoplates. The nanostructures may be formed, for example, of silver or gold reduced with citric acid or another reducing agent, or silver or gold nanostructures obtained by laser ablation or another suitable method. Preferably, the SERS substrate comprises citrate-reduced silver nanoparticles (c-AgNPs). The nanoparticles may be, for example, in an aqueous dispersion or placed on paper.

[0041] Said citrate-reduced silver nanoparticles (c-AgNPs) can be synthesized using the Lee-Meisel method (see reference

[11] ) by dissolving AgNO3 in deionized water (DI), boiling it under magnetic stirring, and then adding a drop of trisodium citrate solution. In a preferred realization, the SERS substrate, preferably c-AgNPs, is mixed with the sample of interest in a ratio comprised between 1:1 and 1:9, preferably in a 1:1 ratio.

[0042] In a further preferred embodiment, the method comprises a sample preparation step prior to step a). Said sample preparation step may comprise a deproteinization step, i.e. a partial or total reduction of the protein content present in the sample. Deproteinization can be carried out using any known method, for example using a centrifugal filter.

[0043] For example, a sample solution is added to a filter and centrifuged to obtain a filtered sample. When using spherical metal nanoparticles as SERS substrates, deproteinization is necessary to form nanoparticle clusters with the plasmonic properties required to achieve a strong SERS effect, because proteins hinder the aggregation of nanoparticles, and deproteinization enhances the effect of SERS.

[0044] The internal standard has formula (I) as described above. In such formula (I), preferably, R1 and R2 are linked to form a fused aromatic ring, particularly phenyl, which is optionally substituted with one or more substituents, preferably substituted with NH2. The substituents may be at any suitable position on the ring, but preferably one substituent is at position 5.

[0045] R3 is preferably H.

[0046] In a preferred embodiment, the internal standard is 5-amino-2-mercaptobenzimidazole (5A2MBI).

[0047] Compounds of formula (I) or (II) are commercially available or can be obtained using conventional synthetic methods according to general knowledge in the art.

[0048] The internal standard is added to the sample in step a) at a concentration that varies depending on the SERS substrate and the characteristics of the substrate to be analyzed. A person skilled in the art can determine the appropriate concentration based on general knowledge in the art. In one embodiment, the concentration of the internal standard is comprised between 2.5 nM and 25 μM, preferably 0.25 μM.

[0049] In a further preferred embodiment of the invention, after step b), the sample together with the SERS substrate is placed on a support, preferably in the form of one or more drops, and dried until completely dry (dry). Preferably, the sample is placed on the support in the form of a drop and the laser measurement is performed on the peripheral ring (coffee ring) of the dried drop. The term "coffee ring" means an area of ​​the dried drop in which the deposited nanoparticles have a higher density compared to the rest of the dried drop ("coffee ring effect"). Preferably, said drop has a volume of more than 1 μL, for example comprised between 30 and 70 μL, preferably 50 μL. The support may be formed, for example, of glass, quartz, calcium fluoride, steel or aluminum. Preferably, said support is selected from calcium fluoride plates and glass plates, which are preferably coated with one layer of aluminum or one layer of aluminum and one layer of parafilm. This last embodiment is particularly advantageous, since it avoids spectral interferences by Raman bands or substrate fluorescence, and moreover, the hydrophobic properties of the parafilm prevent the diffusion of the liquid sample, ensuring the formation of thick, hemispherical substrate droplets.

[0050] In point c), a laser may be used at any wavelength. The wavelength can be selected by the expert in the art depending on the substrate to be analyzed. Preferably, a laser is used at a wavelength comprised between 600 and 850 nm, more preferably at a wavelength of about 785 nm. Any laser device known in the art may be used.

[0051] In an exemplary embodiment of the invention, in step c) the laser is used with a power of 1% (10 mV), an exposure time of 10 seconds and / or a number of accumulations equal to 1. Said power is advantageous since it allows to avoid damage to the sample.

[0052] In a preferred embodiment of the present invention, the amount of EG in a sample is determined by analyzing the SERS spectrum obtained in step c) of the method and detecting the presence of EG at 484 cm -1 The characteristic spectral band of EG at 391-393 cm -1In another embodiment, the characteristic spectral bands of EG are compared to the characteristic spectral bands of other compounds of formula (I) or (II) according to what is known in the art. By comparing the bands, the amount of EG can be determined according to methods known in the art.

[0053] Preferably, this method is used with samples containing EG concentrations below 20 μM, preferably below 10 μM.

[0054] Unless otherwise specified herein, the SERS method can be performed as known in the art, see, e.g., references [9, 12].

[0055] The present invention also provides the use of the compound of formula (I) or (II), preferably 5A2MBI, as an internal standard in the SERS method for measuring the concentration of EG in a sample.Those skilled in the art can use the compound of formula (I) or (II), preferably 5A2MBI, as an internal standard in the SERS method to obtain a calibration curve that allows the determination of the concentration of EG in a sample.

[0056] The method of the present invention can be advantageously applied in the cosmetics, food, nutraceutical and pharmaceutical industries, as well as in biotechnology, bioengineering and clinical diagnostics.

[0057] In an exemplary embodiment of the present invention, a method for determining the amount of ergothioneine (EG) in a sample using surface-enhanced Raman scattering (SERS) spectroscopy includes adding an internal standard, such as 5-amino-2-mercaptobenzimidazole (5A2MBI), to a sample (step a). Then, a SERS substrate is added to the sample containing IS obtained in (a) (step b). The sample obtained in (b) is then exposed to a laser with a wavelength of 785 nm to generate a SERS spectrum (step c).

[0058] In a further exemplary embodiment of the present invention, the IS is 5A2MBI and is added to the sample at a concentration of 0.25 μM and then mixed with the c-AgNP substrate. The concentration of IS 5A2MBI is typically determined based on the intensity value of the internal standard band, which should be lower than that of ergothioneine.

[0059] In a further exemplary embodiment of the present invention, the SERS spectrum of the sample in (c) is obtained using a Raman spectrometer, for example a portable i-Raman plus spectrometer equipped with a 785 nm laser (power output 400 mV) and coupled to a microscope, such as an Olympus miniature microscope with a 20X objective.

[0060] In a further exemplary embodiment of the invention, the SERS spectrum is recorded from 62 to 3202 cm using software such as BWSpec™ version 4.03_23_c. -1 The wavelength range is carried out in the range of 100 nm to 150 nm. The software BWSpec™ makes it possible to obtain a background (dark) signal before data collection and to subtract it from the collected data. Furthermore, the SERS spectrum recording of the sample may include a step of wavelength calibration, which is verified by recording the spectrum of at least one standard reference, such as the spectra of paracetamol and silicon, before and during each recording run.

[0061] In a further exemplary embodiment of the present invention, the method for determining and quantifying the presence of ergothioneine in a sample comprises analyzing the SERS spectrum obtained in (c) and detecting the 484 cm characteristic of ergothioneine. -1 The spectral band of 391-393 cm -1 Compare the spectral bands.

[0062] The invention will now be illustrated by means of examples. EXAMPLES

[0063] Materials and Methods The method is subdivided into a series of very simple steps described as follows: 1. Obtaining a sample of a biological fluid; 2. Adding IS, 5A2MBI to the sample; 3. Combining the sample with the SERS substrate; 4. Exposing the combined sample and SERS substrate to 785 nm laser radiation to generate a SERS spectrum; 5. Analyze the SERS spectrum to find the 484 cm characteristic of ergothioneine. -1 The spectral band of 391-393 cm -1 determining the presence of ergothioneine in the serum sample by comparing the bands with those of

[0064] In step 2, 0.25 μM 5A2MBI is used with the c-AgNP substrate. The concentration determination is based on the intensity of the IS band, which should be lower than that of EG. Normalization with high IS band intensity values ​​may result in an underestimation of the actual measurements.

[0065] Ultrapure water (DI) with a resistivity of 18.2 MΩcm at 25 °C was used throughout the experiment, which was obtained using a Millipore Milli-Q system (Merck, Germany). The buffer solution was prepared by dissolving a tablet of PBS in 200 mL of DI water under magnetic stirring (20 min). L-(+)-ergothioneine (EG) stock solution (10 mM) was prepared by dissolving 2.3 mg of EG in 1 mL of buffer. Adenine solution was prepared by dissolving the powder in 1 M NaOH to obtain a 5 mM solution and subsequently diluting with buffer to a final concentration of 10 μM. A 10 mM stock solution of 5-amino-2-mercaptobenzimidazole (5-A-2MBI) was prepared by dissolving 1.65 mg of powder in 1 mL of methanol. After this step, it was possible to mix the stock solution with buffer to prepare intermediate dilutions before adding to the samples.

[0066] Citrate-reduced silver nanoparticles (c-AgNPs) were synthesized using the Lee-Meisel method

[11] . Briefly, 45 mg of AgNO3 was dissolved in 250 mL of DI water and heated to boiling under magnetic stirring. Subsequently, 5 mL of 1% sodium citrate tribasic solution was added dropwise to the boiling solution. The solution was continued to be heated with stirring for 1 h under complete dark conditions. As a result, a greenish-gray solution was obtained. c-AgNPs were stable for several months when stored at room temperature in the dark. After each preparation, all colloids were characterized by UV-visible extinction spectroscopy using a UV-visible spectrometer (Cary60, Agilent Technology). The extinction band maxima was between 405 and 410 nm, corresponding to an average particle size of 50 nm.

[0067] To evaluate the activity and investigate the batch-to-batch variability of c-AgNPs, a series of SERS measurements were performed using adenine as a reference analyte

[12] . This metabolite has a high affinity for the nanoparticle surface, resulting in a strong and well-defined spectrum, and is also light- and temperature-insensitive, has low toxicity, and is inexpensive.

[0068] SERS equipment SERS spectra were collected at room temperature (22 ± 0.5 °C) using a portable i-Raman plus spectrometer (B&W Tek, Plainsboro, NJ, USA) equipped with a 785 nm laser (power 400 mV) and coupled to a compact microscope with an Olympus 20X objective lens, spot size 108 μm, NA 0.40. Spectral acquisition was performed using BWSpec™ version 4.03_23_c (B&W Tek, Newark, DE, USA) software for the Raman shift range 62–3202 cm. -1 The BWSpec™ software allowed for the collection of a background signal (dark) before data acquisition and for its subtraction from the collected data. Wavenumber calibration was checked before and during each spectral acquisition run by collecting spectra of paracetamol and silicone as standard references.

[0069] result Example 1 The sample preparation is shown in Figure 1. Starting with a volume of 1 mL, the c-AgNPs were concentrated 10-fold in terms of volume in a tabletop centrifuge (15 min x 11337 g). The supernatant (900 μl) was removed and the remaining pellet of nanoparticles was resuspended in 100 μl. At this point, 5 μL of concentrated nanoparticles were distributed into different PCR tubes of 500 μl, one for each sample, and mixed with 5 μL of sample (ratio 1:1). A drop of 5 μL was deposited on slides made of CaF2 or glass and covered with aluminum foil. During this stage of the experiment, it has been shown that the nature of the substrate does not affect the SERS signal. Glass microslides covered with aluminum foil are cheaper and easier to use when compared to CaF2 slides, and for this reason were chosen for subsequent experiments. The droplets on the slide were left to dry for 20 min at room temperature, after which measurements were taken in the peripheral ring of the dried droplet, a distinct area where the analyte signal was stronger, called the "coffee ring". To reduce the influence of variations in the matrix, five measurements were taken per droplet, with a laser power of 1% (10 mV), exposure time of 10 s, and number of integrations equal to 1 to avoid sample damage.

[0070] The results of two experiments using c-AgNPs mixed with the sample of interest and then dried on the support are described in Figure 2. The changes introduced by the internal standard normalization were clear, with little variation in the values ​​for each concentration point, and a linear relationship was evident between the normalized intensity and the EG concentration. Figure 3 describes the complete calibration curve ranging between 1 and 10 μM with the respective figures of merit required for the validation of the calibration model to evaluate the predictive ability and define the quality of the analytical method. The adjusted R-squared value of 0.98 and the narrow bands of error and prediction indicate that the model is good. The limit of detection (LOD), defined as the lowest concentration of analyte in the sample that can be consistently detected with a stated confidence level (typically 95%), was 1.4 μM, and the limit of quantification (LOQ), defined as the lowest concentration of analyte that can be quantitatively detected with acceptable accuracy and precision (10% error), was 2.7 μM.

[0071] Example 2 The sample preparation is shown in Figure 4. 25 μL of human serum enriched with various EG concentrations (Human Serum; Sigma Aldrich; product no. H4522) was mixed with 25 μL of a colloidal dispersion of c-Ag (c-AgNPs) in a 0.5 mL PCR tube. Immediately after mixing, a color change was observed, indicating partial aggregation of the nanoparticles by adsorption of the analyte on the metal surface. The resulting 50 μL droplet was quickly placed under the microscope objective on a glass microslide (25 × 75 mm) pre-covered with aluminum foil and parafilm, mounted on a portable microscope stage. This setup was chosen to avoid spectral interference from Raman bands or fluorescence from the substrate. Furthermore, the hydrophobic properties of the parafilm prevented the diffusion of the liquid sample, ensuring that the droplets were thick and hemispherical. The use of a droplet volume of 50 μL was chosen solely for practical reasons, as it is easy to handle, but if necessary, the volume can be reduced to 30 μL.

[0072] Sample preparation included a step of deproteinization using a centrifugal filter (3 kDa Vivaspin® 0.5 mL, Sartorius, UK). The filter was washed by centrifugation (11337 g × 15 min, 2 times) with DI water before use, after this procedure the sample solution was added and centrifuged (2 cycles of 20 min × 8117 g) to obtain the filtered sample at the bottom of the filter eppendorf. The filtered sample was used for the measurements and was kept at low temperature (4 °C) to avoid degradation. The effect of filtration on the spectra suggests that proteins present in serum (collectively referred to as serum proteins) prevent surface enhancement from occurring and therefore the observation of SERS spectra

[13] , an effect likely due to the lack of nanoparticle aggregation in the presence of serum proteins. When using spherical metal nanoparticles as SERS substrates, at least partial aggregation is necessary to form nanoparticle clusters with suitable plasmonic properties to obtain a strong SERS effect. Aqueous metal colloids are stabilized by electrostatic repulsion that breaks the nanoparticles apart and thus prevents their aggregation when colliding with each other. The stabilizing surface charge mostly comes from charged species directly adsorbed on the nanoparticle surface (like citrate ions for c-AuNPs and c-AgNPs or chloride ions for h-AgNPs). However, when a high electrolyte content is introduced, such as serum or plasma, the stabilizing surface charge is "shielded" by the ions in the solution, causing rapid aggregation of the nanoparticles. However, serum proteins (mostly albumin) are known to readily adsorb to the metal nanoparticle surface and form a layer called the "protein corona", which establishes steric repulsion between the nanoparticles that prevents them from colliding with each other and thus avoids aggregation. The results are depicted in Figure 5. The changes introduced by normalization with the internal standard were clear, with little variation in the values ​​for each point concentration and a linear relationship between normalized intensity and EG concentration was evident.

[0073] Figure 6 lists the complete calibration curve ranging between 0.2 and 2 μM with the respective parameters required for validation of the calibration curve model in order to assess the predictive ability and define the quality of the analytical method. The coefficient of determination R 2 The value of 0.97 and the narrow confidence and prediction bands indicate that the error of this model is low. The limit of detection (LOD), defined as the lowest concentration of analyte in the sample that can be consistently detected with a stated confidence level (typically 95%), was 0.5 μM, and the limit of quantification (LOQ), defined as the lowest concentration of analyte that can be quantitatively detected with acceptable accuracy and precision (10% error), was 0.9 μM.

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Claims

1. The process is as follows: a) adding at least one internal standard (IS) to a sample to obtain a sample containing at least one IS; b) adding at least one SERS substrate to the sample containing at least one IS obtained in a); c) exposing the sample together with the SERS substrate obtained in step b) to a laser to generate a SERS spectrum; 1. A method for detecting and / or determining the amount of ergothioneine (EG) in a sample using surface-enhanced Raman spectroscopy (SERS), comprising: The internal standard has the following formula (I): 【Chemistry 1】 (In the formula, R1 and R2 are each independently C 1～3 -Alkyl, NH 2 , N.H.C.O. 1～3 Alkyl, NH(C 1～3 -alkyl), N(C 1～3- Alkyl) 2 , halogen, aryl, and heteroaryl; or R1 and R2 are combined, each of which is optionally C 1～3 -Alkyl, NH 2 , N.H.C.O. 1～3 Alkyl, NH(C 1～3 -alkyl), N(C 1～3 -alkyl) 2 , halogen, NO 2 , CN, and forming a fused aromatic or heteroaromatic ring selected from phenyl, pyrrole, imidazole, pyridine, pyrimidine, substituted with one or more substituents selected from - R3 is H, CH 3 , or NH 2 (It is) The method of claim 1, characterized by the fact that

2. The internal standard has the formula (I), in which R1 and R2 are attached to a fused aromatic ring, in particular a fused aromatic ring, optionally substituted with one or more substituents, preferably NH 2 4. The method of claim 1, wherein R is substituted with R, forming a phenyl, and / or R is H.

3. The internal standard has formula (II): 【Chemistry 2】 (In the formula, R3 is H, CH 3 , or NH 2 is preferably H, R4 is NH 2 , NO 2 , H, or halogen, preferably NH 2 3. The method of claim 1 or 2, wherein

4. 4. The method of claim 1, wherein the internal standard is 5-amino-2-mercaptobenzimidazole (5A2MBI).

5. 5. The method according to any one of claims 1 to 4, wherein in step a) the internal standard is added at a concentration comprised between 2.5 nM and 25 μM, preferably at a concentration of 0.25 μM.

6. 6. The method according to claim 1, wherein in step c) the laser used has a wavelength comprised between 600 nm and 850 nm, preferably 785 nm.

7. The presence and / or amount of EG in the sample is -1 7. The method according to claim 1 , further comprising a further step d) of determining the characteristic spectral bands of EG at 20° C. by comparing the characteristic spectral bands of the internal standard with the characteristic spectral bands of the internal standard.

8. The internal standard is 5A2MBI, whose characteristic spectral band is 391-393 cm -1 The method according to claim 7, wherein

9. 9. The method of claim 1, wherein the SERS substrate comprises at least one nanostructure selected from spheroidal nanoparticles, nanorods, nanostars, and nanoplates.

10. 10. The method according to any one of claims 1 to 9, wherein the SERS substrate comprises at least one nanostructure of silver or gold reduced with citric acid or another reducing agent, or at least one nanostructure of silver or gold obtained by laser ablation.

11. 11. The method of claim 1, wherein the SERS substrate comprises citrate-reduced silver nanoparticles (c-AgNPs).

12. 12. The method according to any one of claims 1 to 11, wherein the sample is selected from an isolated biological sample, such as a body fluid, a food product, and a cosmetic product.

13. The method according to any one of claims 1 to 12, wherein the sample is a body fluid, preferably serum.

14. 14. The method according to any one of claims 1 to 13, wherein the method further comprises a step of deproteinization of the sample prior to step a).

15. 15. The method according to any one of claims 1 to 14, wherein after step b) the sample together with the SERS substrate is placed, preferably in the form of one or more drops, on a support and left to dry until dry.

16. 16. The method according to claim 15, wherein the support is selected from calcium fluoride plates and glass plates, preferably coated with at least one layer of aluminium or at least one layer of aluminium and at least one layer of parafilm.

17. Formula (I) 【Chemistry 3】 (In the formula, R1 and R2 are each independently C 1～3 -Alkyl, NH 2 , N.H.C.O. 1～3 Alkyl, NH(C 1～3 -alkyl), N(C 1～3- Alkyl) 2 , halogen, aryl, and heteroaryl; or R1 and R2 are combined, each of which is optionally C 1～3 -Alkyl, NH 2 , N.H.C.O. 1～3 Alkyl, NH(C 1～3 -alkyl), N(C 1～3 -alkyl) 2 , halogen, NO 2 , CN, and forming a fused aromatic or heteroaromatic ring selected from phenyl, pyrrole, imidazole, pyridine, pyrimidine, substituted with one or more substituents selected from - R3 is H, CH 3 , or NH 2 (It is) Use of this compound as an internal standard in a SERS method for measuring the concentration of EG in a sample.

18. 18. The use according to claim 17, wherein the compound of formula (I) is 5-amino-2-mercaptobenzimidazole (5A2MBI).

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