A method for producing surface-enhanced Raman spectroscopic labels

Abstract

This invention relates to the field of methods for producing surface enhanced Raman spectroscopy (SERS) tags. The production method according to the invention is reproducible and versatile and allows for the production of high quantities of SERS tags in a convenient manner, characterized by a narrow size distribution and a high proportion of small particles. The SERS tags produced by the inventive production method described herein exhibit a wider range of SERS responses.

Description

A method for producing surface-enhanced Raman spectroscopy labels Field of invention This invention relates to the field of methods for producing surface-enhanced Raman spectroscopy (SERS) tags. The production method according to the invention is reproducible and versatile and allows for the production of large quantities of SERS tags in a convenient manner, characterized by a narrow size distribution and a high proportion of small particles. The SERS tags produced by the inventive production method described herein provide a wider range of SERS responses. Background to this invention Surface-enhanced Raman spectroscopy (SERS) tags are useful for a variety of applications, including product labeling for identification and authentication, and high-throughput multiplex screening in microarray technology, diagnostics, and bioimaging. SERS tags are a collection of nanoparticles that present a plasmonic surface and adsorb Raman-active reporter molecules onto their surface. The nanoparticles that present a plasmonic surface are responsible for generating the electric field required for Raman enhancement, while the Raman-active reporter molecule provides the unique vibrational fingerprint of the SERS tag. Typically, the particles provide an external coating layer that a) isolates the SERS tag from the external environment, preventing the release of Raman-active reporter molecules from the SERS tag and protecting the SERS tag from external environmental contaminants that may increase vibrational noise, b) increases the colloidal stability of the SERS tag, and c) provides a suitable surface for further chemical functionalization.So far, polymers and silica have been used as outer coating layers. Due to the strong dependence of plasmonic properties on the aggregation states of nanoparticles, it is highly desirable to produce SERS tags with a high number of low-number particles to obtain a larger set of SERS responses. SERS tags with a high number of low-number particles were produced either through post-synthesis sorting techniques or through controlled aggregation synthesis processes. For example, field-current fractionation applied to a mixture containing single nanoparticles and nanoparticle particles from dimers to octamers resulted in a particle enriched in dimer (10%), trimer (21%), and tetramer (13%) SERS tags, yet containing a high percentage of single nanoparticles (52%) (J. Am. Chem. Soc. 2010, 132, 10903–10910). A mixture containing nanoparticles and single particles from dimers to duomers was enriched in dimer (52%) and trimer (32%) SERS tags using a centrifugal post-synthesis sorting method in a high density gradient medium, such as an aqueous iodixanol density gradient (US9802818B2). In addition to being time-consuming and expensive, post-synthesis sorting techniques require the use of harsh conditions (e.g., high-viscosity reagents), which leads to instability of uncoated SERS tags. Therefore, post-synthesis sorting techniques are only compatible with silica- or polymer-coated SERS tags. Therefore, controlled aggregation synthetic processes have a great advantage for producing SERS tags with a narrow size distribution and a high proportion of small-number particles.Solid-substrate-assisted aggregation allowed the production of SERS tags with a narrow size distribution. Ruan et al. (Adv. Optical Mater. 2014, 2, 65 - 73) described the synthesis of asymmetric core-functional SERS tags with a functional number of 2±18 gold nanospheres with an average diameter of 24 nm per gold nanosphere core with an average diameter of 180 nm. The synthesis involved adsorption of cetyltrimethylammonium bromide-stabilized gold nanoparticle cores onto an indium tin oxide-coated glass slide or silicon wafer, followed by immersion of the functionalized solid substrate in a solution of 4-aminothiophenol in water / acetonitrile to adsorb the Raman-active reporter molecule onto the surface of the gold nanoparticles and subsequent immersion for 1 h in a suspension of gold nanoparticles with an average diameter of 24 nm. The aggregation state of SERS tags can be controlled by controlling the immersion time of the functionalized solid substrate in the suspension of gold nanoparticle functions and / or reducing the concentration of gold nanoparticle functions in said suspension.The method described by Ruan et al. relies on the use of the surfactant cetyltrimethylammonium bromide to immobilize the gold colloid, which significantly reduces the surface area available for adsorption of the Raman-active reporter molecule on the gold nanoparticle core and, consequently, reduces the intensity of the SERS signal provided by the SERS tags. To attach the gold nanoparticle functions to the gold nanoparticle core, the aggregation method uses Raman-active reporter molecules that present two functionalities with combined affinity for the gold surface. Therefore, this method is only applicable to the production of SERS tags that have Raman-active reporter molecules that present two functionalities with combined affinity for the gold surface. This method represents a high limitation of Raman-active reporter molecules for use as SERS tag fingerprints. Furthermore, the method involves long reaction times and is not suitable for the production of large quantities of SERS tags. Another method for the solid-based assembly of SERS tags with a narrow size distribution was described by Yoon et al. (ACS Nano 2012, 8, 7199–7208). This method is based on the size-dependent repulsion propensities of gold nanoparticles adsorbed on amino-functionalized glass slides and the use of alkane dithiols to attach gold nanoparticle functionalities to the gold nanoparticle core. SERS tags with 3±13 gold nanoparticle functionalities with an average diameter of 13 nm were produced per gold nanoparticle core with an average diameter of 51 nm. The method developed by Yoon et al. appears to enable incorporation of any Raman-active reporter molecule into the SERS tag. However, the method has limitations in terms of the gold nanoparticle sizes that can be used and placed on the gold nanoparticle core accessible to the Raman-active reporter molecules. Furthermore, this method requires long reaction times and is not suitable for the production of sufficiently high amounts of SERS labels. Therefore, there is a need for reproducible, cost-effective, and versatile methods for producing SERS tags that allow for the convenient production of large quantities of SERS tags characterized by a narrow size distribution and a high proportion of low-number particles. SERS tags with a high number of low-number clusters are highly desirable to obtain a larger set of SERS responses. Summary of the invention Accordingly, the object of the present invention is to provide a versatile, cost-effective and reproducible method for producing SERS tags, which allows for the production of large quantities of SERS tags in a convenient manner, characterized by a narrow size distribution and a high low-number aggregation ratio. This is achieved by a method for producing surface-enhanced Raman spectroscopy (SERS) tags, preferably SERS tags for use as a security element, comprising the following steps: A) Preparation of the first colloid, consisting essentially of nanoparticles with a plasmonic surface that are approximately the same size and are dispersed in an aqueous solvent, and a stabilizing agent is adsorbed on the surface of said nanoparticles and has a potential value of ζ less than or equal to -25 mV. b) Preparation of the second colloid consisting essentially of surface plasmonic nanoparticles of approximately uniform size dispersed in an aqueous solvent. Raman-active reporter molecules and a stabilizing agent are adsorbed on the surface of said nanoparticles and have a potential ζ value less than or equal to -25 mV. c) To create the third colloid, the first colloid must be combined with the second colloid such that the ratio between the number of nanoparticles of the first colloid and the number of nanoparticles of the second colloid is between about 25:1 and 1:1, preferably from about 1:5 to 1:1, more preferably from 4:1 to about 1:3. d) Creating nanoparticle aggregation by any of the steps (d1-d3) or a combination of them: d1) The third colloid composition obtained in step c) with a pH between 2.2 and the lowest pH value at which the Raman active reporter molecules have a net electrical charge between 0 and 0.3. d2) Adding a salt solution, preferably a mineral salt solution, to the third colloid obtained in step c). d3) adding a water-miscible solvent to the third colloid obtained in step c) and e) Stop the gathering. Preferably, step b) in the method claimed and described herein comprises the following steps, performed in order from b1) to b3): b1) preparing a colloid consisting essentially of nanoparticles with a plasmonic surface that are approximately the same size and are dispersed in an aqueous solvent and a stabilizing agent is adsorbed on the surface of said nanoparticles and has a potential value ζ less than or equal to -25 mV; b2) adjusting the pH of the colloid to the lowest pH value at which the Raman-active reporter molecules adsorbed on the surface of the nanoparticles have no net electrical charge, while the ζ potential value is less than or equal to -25 mV, preferably less than -40 mV, and b3) Adding a solution of Raman-active reporter molecules in a solvent to the colloid obtained in step b2), while the ζ potential value is less than or equal to -25 mV. In another preferred embodiment, steps c) and d) are carried out simultaneously in a continuous flow system. In another preferred embodiment, the size of the nanoparticles in the first colloid is different from the size of the nanoparticles in the second colloid. As shown in Examples E1-E14, the method claimed herein allows for the incorporation of a variety of Raman active reporter molecules into the SERS tag and is not limited to a combination of specific sizes of nanoparticles. It also enables the synthesis of SERS tags composed of nanoparticles having the same size as confirmed for example by Examples E1-E8 and E12-E14. In addition, the manufacturing method claimed and described herein provides SERS tags with a narrow size distribution as shown for example by Figure 3A and Figure 3B and a high proportion of small number particles as shown for example by Figure 3A and Figure 3B, and has a relatively shorter reaction time than methods known in the art. Brief description of the shapes Figure 1A schematically illustrates a method of producing SERS tags in accordance with the present invention. The method of production comprises combining a second colloid (120a) consisting of surface plasmonic nanoparticles of approximately the same size (121a) dispersed in an aqueous solvent (not shown) in which the nanoparticles have adsorbed Raman-active reporter molecules on their surface (a122). A stabilizing agent (not shown) with a first colloid consisting essentially of surface plasmonic nanoparticles of approximately the same size (130a) dispersed in an aqueous solvent (not shown) in which the nanoparticles have adsorbed a stabilizing agent (not shown) on their surface to prevent the aggregation of the nanoparticles to provide SERS tags (140a). Figure 1b schematically illustrates an embodiment of a method for producing SERS tags according to the present invention. In this particular method of production, a continuous flow reactor comprises two pressure vessels (111b, 112b), a tee (113b) and a collection vessel (114b), wherein each of the two pressure vessels (111b, 112b) is connected via a tube to the tee (113b), which in turn is connected via a tube to the collection vessel (114b). The production method comprises simultaneous mixing and blending at a pH between 2.2 and a minimum pH value at which Raman active reporter molecules have a net electrical charge between 0 and 0.3 in the tee (113b) to collection vessel (114b) connection tube of the continuous flow reactor. The first colloid is provided by a pressurized reservoir (112b) and is essentially composed of surface plasmonic nanoparticles of approximately the same size (130b) dispersed in an aqueous solvent (not shown) in which said nanoparticles have adsorbed a stabilizing agent (not shown) on their surface.The second colloid (120b) is provided by the pressurized reservoir (111b) and consists essentially of surface plasmonic nanoparticles of approximately the same size (121b) dispersed in an aqueous solvent (not shown) in which the nanoparticles have adsorbed on their surface Raman-active reporter molecules (122b) and a stabilizing agent (not shown). The combination at a pH between 2.2 and a minimum pH value at which the Raman-active reporter molecules have a net electrical charge between 0 and 0.3 causes the nanoparticles to aggregate. The aggregation step is stopped in the collection reservoir (114b) thereby providing the target SERS labels (140b). Figure 1c schematically illustrates an embodiment of a method for producing SERS tags in accordance with the present invention. In this particular method of production, a continuous flow reactor comprises three pressure vessels (111c, 112c, 150c), a tee (113c) and a collection vessel (114c). Each of the two pressure vessels (111c, 112c) is connected via a tube to the tee (113c), which in turn is connected via a tube to the collection vessel (114c). The production method involves simultaneous mixing and blending at a pH between about 2.2 and a minimum pH value at which the Raman active reporter molecules have a net electrical charge of between 0 and 0.3 in the tee (113c) to collection vessel (114c) connection tube of the continuous flow reactor. The first colloid, provided by the pressurized reservoir (112c), is essentially composed of surface plasmonic nanoparticles of approximately the same size (130c) dispersed in an aqueous solvent (shown) in which the nanoparticles have adsorbed a stabilizing agent on their surface (not shown).The second colloid (120c) is provided by a pressurized reservoir (111c) and consists essentially of surface plasmonic nanoparticles of approximately the same size as (121c) dispersed in an aqueous solvent, in which the nanoparticles have adsorbed on their surface Raman-active reporter molecules (122c) and a stabilizing agent (not shown). The combination causes the nanoparticles to aggregate at a pH between 2.2 and a minimum pH value at which the Raman-active reporter molecules have a net electrical charge between 0 and 0.3. The aggregation step is terminated by: introducing water stored in the pressure tank (150c) into the pipe leading to the collection tank (114c) to dilute the colloid, introducing a base solution stored in the pressure tank (150c) into the pipe leading to the collection tank (114c), introducing a polymer stored in the pressure tank (150c) into the pipe leading to the collection tank (114c), or introducing a dielectric precursor stored in the pressure tank (150c) into the pipe leading to the collection tank (114c), thereby providing the target SERS labels (140c). Figure 2a-c shows the variation of SERS signal intensity with the accumulation reaction time: Figure 2a shows the variation of SERS intensity with the accumulation reaction time for SERS tags produced according to Example E1. Figure 2b shows the variation of SERS intensity with the accumulation reaction time for SERS tags produced according to Example E14. Figure 2c shows the variation of SERS intensity with the accumulation reaction time for SERS tags produced according to Example E13. The horizontal axis corresponds to the accumulation reaction time in seconds and the vertical axis corresponds to the SERS response with 785 nm excitation. The reported SERS intensity was measured with 100 ms integration time every 1 s at the given gold concentration and this signal intensity is related to samples diluted to 12.5 μg gold / mL and scanned at 1 s integration. Figure 3A-B presents SEM images taken of SERS tags containing nanoparticles of different sizes, produced according to Examples E9 (Figure 3A) and 10 (Figure 3B). As confirmed by the SEM images, the SERS tags produced according to the present invention are enriched in low-number particles such as dimers, trimers, and tetramers and have a narrow distribution. Figure 4 is a graph showing the ζ potential value of the first colloid A2 (drawn as solid circles) and the second colloids D2 (drawn as solid triangles), D5 (drawn as solid diamonds), and D6 (drawn as solid squares) when the following colloids are titrated with 1N HCl aqueous solution or 1N NaOH aqueous solution. The horizontal axis corresponds to the pH value and the vertical axis corresponds to the ζ-potential value (mV). The conditions under which aggregation was observed (i.e., the colloids are unstable) are circles. As shown in Figure 4, the types of colloids characterized by a ζ-potential value of less than -25 mV are stable. Such colloids can be used as the first and second colloids, respectively, in the production method according to the present invention. Figure 5 shows the development of the SERS signal intensity of aggregated colloids in a flow system. Each measurement was made at 1-meter intervals along a 7-meter-long transparent FEP tube. The data shown by the diamonds connected by a solid line were applied to the SERS labels produced by Example E8. The data shown by the circles connected by a dotted line were applied to the SERS labels produced by Example E11. Description with details Definitions The following definitions are used to interpret the meaning of terms discussed in this specification and recited in the claims. As used here, the definite article "one" indicates one as well as more than one and does not necessarily limit its referent to the singular. As used herein, the term "at least" is used to define one or more than one, for example one or two or three. The term "comprising" as used herein is non-exclusive and open-ended. Thus, for example, a solution comprising a compound A may include compounds other than A. However, the term "comprising" also includes, as a specific embodiment thereof, the more restrictive meanings of "consisting essentially of" and "consisting of", so for example, "a solution comprising A, B and C optionally" may also consist (essentially) of A and B, or (essentially) of A, B and C. Where this description refers to "preferred" embodiments / features, the combination of the "preferred" embodiments / features is deemed to be disclosed as long as the combination is technically meaningful. As used herein, the term "range" means that the amount or amount of interest may be the specific value specified or another value within the same range. In general, the term "range" indicating a specific value indicates a range of about ± 5% of that value. For example, the term "about 100" indicates a range of 100 ± 5, i.e., a range of 95 to 105. Preferably, the range indicated by the term "range" indicates a range of about ± 3% of the value, more preferably ± 1%. In general, when the term "range" is used, it can be expected that similar results or effects in accordance with the invention will be obtained within ± 5% of the indicated value. Surprisingly, it was found that the method used to produce surface-enhanced Raman spectroscopy (SERS) tags, preferably SERS tags as a security element, involves the following steps: a) Preparing a first colloid consisting of nanoparticles with a plasmonic surface that are approximately the same size and are dispersed in an aqueous solvent and a stabilizing agent is adsorbed on the surface of said nanoparticles and has a potential value ζ less than or equal to -25 mV; b) Preparation of a second colloid consisting essentially of surface plasmonic nanoparticles of approximately the same size dispersed in an aqueous solvent, with Raman-active reporter molecules and a stabilizing agent adsorbed on the surface of said nanoparticles, and having a potential ζ value less than or equal to -25 mV. c) To create the third colloid, the first colloid must be combined with the second colloid such that the ratio between the number of nanoparticles of the first colloid and the number of nanoparticles of the second colloid is between about 1:25 and 1:1, preferably from about 1:5 to 1:1, more preferably from 1:4 to about 1:3. d) Creating nanoparticle aggregation by any of the steps (d1-d3) or a combination of them: d1) The third colloid composition obtained in step c) with a pH between 2.2 and the lowest pH value at which the Raman active reporter molecules have a net electrical charge between 0 and 0.3. d2) Adding a salt solution, preferably a mineral salt solution, to the third colloid obtained in step c). d3) adding a water-miscible solvent to the third colloid obtained in step c) and e) Stop the gathering; It provides high amounts of SERS tags with a narrow size distribution and a high proportion of low-number clusters in a cost-effective and convenient manner. This method has no limitations in terms of the Raman-active reporter molecule to be used as the SERS tag fingerprint or the size of the surface plasmonic nanoparticles present in the first and second colloids, thus allowing the production of a variety of SERS tags. As is known to those skilled in the art and as used herein, a SERS tag comprises a set of nanoparticles that present a plasmonic surface and Raman-active reporter molecules adsorbed on the surface of the nanoparticles. The nanoparticles that present the plasmonic surface are responsible for generating the electric field required for Raman amplification, while the Raman-active reporter molecules provide the unique vibrational fingerprint of the SERS tag. A SERS tag may further comprise an outer coating layer that isolates the nanoparticles adsorbed on the surface of the Raman-active molecules from the external environment. Thus, the outer coating layer a) isolates the SERS tag from the external environment, thereby preventing the Raman-active reporter molecules from leaving the SERS tag and protects the SERS tag from external environmental contamination that may lead to spurious peaks. b) increases the colloidal stability of the SERS label and c) provides a suitable surface for further chemical application.The outer coating layers consist of silica and polymers such as poly(ethyleneimine) (PEI), poly(styrene-alt-maleic acid), sodium salt (PSMA), poly(diallyldimethylammonium chloride) (PDADMAC). In view of the displayed SERS signal, the SERS labels obtained by the claimed and described production method are particularly useful as a security element for protecting documents and goods against counterfeiting and illegal reproduction. As used herein, the term "security element" designates an element that can be incorporated into a security document or article or used for the purpose of authentication and protection against counterfeiting and illegal reproduction. The security element may be a symbol, image, pattern or graphic element that is printed, coated or sprayed onto a security element or article with an ink, varnish or coating composition containing the SERS labels obtained by the production method according to the present invention. Alternatively, the SERS labels may function as a security element when integrated into the substrate of a security document.The terms “security document” and “security item” refer to a document or item that has a value that makes it potentially vulnerable to attempted forgery or unlawful reproduction and is usually protected by at least one security feature against forgery or fraud. The term “security items” as used herein includes all items that must be protected against forgery and / or unlawful reproduction in order to ensure their contents. Examples of security documents include commercial documents and items of unlimited value. Typical examples of valuable documents include, without limitation: banknotes, certificates, tickets, checks, coupons, tax stamps, tax labels, contracts and the like, identity documents such as passports, ID cards, visas, bank cards, credit cards, transaction cards, access documents, entry tickets and the like.Valuable commercial goods include: packaging materials, especially for cosmetics, food products, pharmaceutical products, alcohol, tobacco products, beverages or food, electrical / electronic products, textiles or jewellery, for example items that need to be protected against counterfeiting and / or illegal reproduction to ensure the contents of the packaging, such as original medicines. Examples of packaging materials include, without limitation: labels, such as authentication trademark labels, tamper evidence labels and seals. In the context of this invention, the term "nanoparticle" is defined as a single particle with a size corresponding to the maximum physical dimensions (such as length, diameter, etc.) in the range of 5±20 nm to 5±160 nm, preferably from 5±40 nm to 5±140 nm. The nanoparticle used in this invention has a plasmonic surface, i.e. the nanoparticle has an outer surface that can enhance the Raman scattering of a Raman active molecule. The outer surface of the nanoparticle is produced from any known SERS enhancing material. Preferably, the SERS enhancing material is selected from: gold (Au), silver (Ag), copper (Cu), aluminum (Al), palladium (Pd), platinum (Pt) or a mixture or alloy thereof and more preferably, from gold (Au). The nanoparticle used in this invention may be solid or hollow and preferably solid. A solid nanoparticle may be made from a single material, i.e. the SERS enhancing material on the outer surface of the nanoparticle, or it may be made from more materials, i.e. the core material(s) of the nanoparticle may be different from the SERS enhancing material on the outer surface of the nanoparticle. A hollow nanoparticle is a nanoparticle whose core is empty space.The nanoparticle may have any shape that can be produced. Preferably, the nanoparticle is a solid gold nanoparticle. Preferably, the nanoparticle has a shape selected from the group consisting of a sphere, a pseudosphere, a rod, a disk, a prism, and a cube. More preferably, it is selected from a sphere and a pseudosphere, and even more preferably, the nanoparticle has a pseudospherical shape. As used herein, the term "nanoparticles of approximately uniform size" means that the size of said nanoparticles is determined by maximum physical dimensions (e.g. length, diameter, etc.) within ± 20 nm, preferably within ± 10 nm of the average size, and for said nanoparticles is consistent with electron microscopy methods, such as transmission electron microscopy (TEM) or scanning electron microscopy (SEM), or, if possible, with the method of Haiss et al. (Anal. Chem. 2007, 79, 4215-4221). The nanoparticles of the first colloid, the second colloid and the colloid provided in step b1) are dispersed in an aqueous solvent. As used herein, the term "aqueous solvent" refers to water and a mixture of water with one or more water-miscible solvents, wherein the water-miscible solvent is preferably selected from the list consisting of: methanol, ethanol, propanol, isopropanol, tetrahydrofuran, N-methyl-2-pyrrolidone (NMP), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), acetone and acetonitrile. The first colloid provided in step a) of this production method, the second colloid provided in step b) of this production method, like the colloid provided in step b1) of this production method, do not contain a polymer or surfactant for stabilizing purposes. Stabilization of colloids with polymers (e.g. polyvinylpyrrolidone) or surfactants (e.g. cetyltrimethylammonium bromide) is well known in the art. However, since polymers and surfactants are added to the colloids before the addition of the Raman-active reporter molecules, they significantly reduce the surface area available on the nanoparticles for the adsorption of the Raman-active reporter molecules and increase the distance between the colloidal particles in an aggregate, which results in the SERS labels showing a lower SERS signal intensity. To overcome these drawbacks, the first colloid, the second colloid, as well as the colloid provided in step b1) are surfactant-free and polymer-free. The stabilizing agent adsorbed on the surface of the first colloid, second colloid and the colloid provided in step b1) nanoparticles is preferably selected from carboxylic acids, carboxylic acid salts, phosphoric acids, phosphoric acid salts, ascorbic acid, ascorbic acid salts and mixtures thereof. To avoid competition between the Raman-active reporter molecules and the stabilizing agent during adsorption of the Raman-active reporter molecules on the surface of the nanoparticles, the stabilizing agent preferably does not contain groups that show affinity for SERS enhancing materials, in particular gold. Examples of such groups include: nitrogen-containing groups, sulfur-containing groups, ethynyl groups, cyano groups and isocyanide groups. As used herein, the term "carboxylic acid" refers to an organic compound containing a carboxyl group (C(═O)OH) and includes monocarboxylic acids (i.e., organic compounds containing a single carboxyl group), such as lactic acid, and polycarboxylic acids (i.e., organic compounds containing two or more carboxyl groups), such as citric acid. As used herein, the term "carboxylic acid salt" refers to the sodium or potassium salt of a carboxylic acid. Preferably, the stabilizing agent is selected from carboxylic acids, carboxylic acid salts, ascorbic acid, ascorbic acid salts, and mixtures thereof. Even more preferably, the stabilizing agent is selected from citric acid, citric acid salts, lactic acid, lactic acid salts, ascorbic acid, ascorbic acid salts, and mixtures thereof. Citric acid salts include monosodium dihydrogen citrate, disodium hydrogen citrate, trisodium citrate, monopotassium dihydrogen citrate, potassium dihydrogen citrate, and tripotassium citrate. Lactic acid salts include sodium lactate and potassium lactate. Ascorbic acid salts include sodium ascorbate and potassium ascorbate. In a preferred embodiment, the stabilizing agent is selected from citric acid, monosodium dihydrogen citrate, disodium hydrogen citrate, trisodium citrate, monopotassium dihydrogen citrate, dipotassium hydrogen citrate, and tripotassium citrate, and mixtures thereof. Advantageously, the claimed and described production method allows the incorporation of any Raman active reporter molecule into a SERS label. Preferred Raman active reporter molecules include fully conjugated (double) molecules consisting of an aryl group substituted with one or more substituents selected from NR1R2, –SH, —≡, —≡N and —N═, preferably selected from –NR1R2 and –SH and / or an N-containing heteroaryl group and / or an S-containing heteroaryl group, wherein the residues R1 and R2 are independently selected from H- and alkyl, preferably from H- and -C4-C1alkyl. As is well known to those skilled in the art of organic chemistry, a fully conjugated molecule is one that has a conjugated electron system that extends throughout the molecule. The conjugated electron system is a system of p orbitals connected by delocalized electrons. As is known to those skilled in the art, an "aryl group" is a group derived from a monocyclic or polycyclic aromatic hydrocarbon compound by removing a hydrogen atom from a ring of carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthryl, and pyrenyl. An S-containing heteroaromatic compound is an aromatic compound containing a sulfur heteroatom as part of a π-conjugated ring system. As part of the π-conjugated ring system, an S-containing heteroaromatic compound may contain one or more nitrogen atoms. Examples of S-containing heteroaryl groups include, but are not limited to, thiophenyl, thiazolyl, isothiazolyl, thiadiazolyl, benzothiophenyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, imidazothiazolyl, and imidazothiadiazolyl. An N-containing heteroaromatic compound is an aromatic compound containing at least one nitrogen heteroatom as part of a π-conjugated ring system. As part of a π-conjugated ring system, an N-containing heteroaromatic compound may contain one or more oxygen atoms. Examples of N-containing heteroaryl groups include, without limitation, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, benzoimidazolyl, indazolyl, benzotriazolyl, pyridinyl, pyrimidinyl, pyridazinyl, triazinyl, quinolinyl, isoquinolinyl, diazanaphthyl, quinalozinyl, cinnolinyl, phthalazinyl, quinoxalinyl, purinyl, aza-phenanthryl, diaza-phenanthryl, aza-anthracenyl, diaza-anthracenyl, aza-pyrenyl, diazapyrenyl, oxazolyl, isoxazolyl, hexadiazolyl, benzoxazolyl, benzisoxazolyl, and benzoxadazolyl. Preferred Raman-active reporter molecules include, but are not limited to: - A fully conjugated compound consisting of an aryl group substituted with one or more substituents selected from —NR1R2, —SH, —≡, —≡N and —N═, preferably —NR1R2 and —SH, and attached directly or through a linker —L1— to an aryl group substituted with one or more substituents selected from the list consisting of an amino group (NH2—), an N-alkyl amino group, an N,N-dialkyl amino group, a thiol group, an ethynyl group, a cyano group and an isocyanide group, an N-containing heteroaryl group, or an S-containing heteroaryl group, wherein The substituents R1 and R2 have the meanings defined herein; The linker -L1- is selected from the following links: —CR8═CR9—, —N=N—, —≡—, —CR10═CR11— o -C6H4—, —CR10═CR11— m -C6H4—, —CR10═CR11— p -C6H4—, —CR10═CR11— o -C6H4—CR12═CR13—, —CR10═CR11— m -C6H4—CR12═CR13—, —CR10═CR11— p -C6H4—CR12═CR13—, —CR14═N—N═CR15—, , , , , , , , , , , , , , , , , , , , , , , , and ; and The substituents R8-R15 are selected from hydrogen, alkyl, alkoxy, alkylthio, formyl, cyano, nitro, halide, hydroxycarbonyl and alkoxycarbonyl. - A fully conjugated compound consisting of an N-containing heteroaryl group attached directly or through an -L1- linker to an N-containing heteroaryl group or an S-containing heteroaryl group. in which The linker -L1- is selected from the following links: —CR8═CR9—, —N=N—, —≡—, —CR10═CR11— o -C6H4—, —CR10═CR11— m -C6H4—, —CR10═CR11— p -C6H4—, —CR10═CR11— o -C6H4—CR12═CR13—, —CR10═CR11— m -C6H4—CR12═CR13—, —CR10═CR11— p -C6H4—CR12═CR13—, —CR14═N—N═CR15—, , , , , , , , , , , , , , , , , , , , , , , , and ; and The substituents R8-R15 are selected from hydrogen, alkyl, alkoxy, alkylthio, formyl, cyano, nitro, halide, hydroxycarbonyl and alkoxycarbonyl. - A fully conjugated compound consisting of an S-containing heteroaryl group attached directly or through an -L1- linker to an S-containing heteroaryl group. in which The linker -L1- is selected from the following links: —CR8═CR9—, —N=N—, —≡—, —CR10═CR11— o -C6H4—, —CR10═CR11— m -C6H4—, —CR10═CR11— p -C6H4—, —CR10═CR11— o -C6H4—CR12═CR13—, —CR10═CR11— m -C6H4—CR12═CR13—, —CR10═CR11— p -C6H4—CR12═CR13—, —CR14═N—N═CR15—, , , , , , , , , , , , , , , , , , , , , , , , and ; and the substituents R8-R15 are selected from hydrogen, alkyl, alkoxy, alkylthio, formyl, cyano, nitro, halide, hydroxycarbonyl and alkoxycarbonyl. and - A fully conjugated compound consisting of an aryl group substituted with one or more, preferably at least two, substituents selected from NR1R2___, —SH, —≡, —≡N and —N═, preferably from NR1R2— and SH—. An N-containing heteroaryl group optionally substituted with one or more substituents selected from NR3R4-, SH—, —≡, —≡N and —N═ ​​or an S-containing heteroaryl group optionally substituted with one or more substituents selected from NR5R6___, SH—, —≡, —≡N and —N═ ​​which is directly bonded to a hydrogen atom and in which the substituents R1 and R2 have the meanings defined herein and the substituents R3-R6 are independently selected from H- and alkyl, preferably from H- and alkyl-C1-C4. The aryl group substituted by one or more substituents selected from NR1R2—, —SH, —≡, —≡N and —N═, preferably from NR1R2— and —SH, may contain one or more further substituents, preferably selected from the group consisting of: hydroxy, alkyl, alkoxy, alkylthio, formyl, nitro, halide, hydroxycarbonyl, alkoxycarbonyl and O-containing heteroaryl groups, and more preferably selected from: alkyl, alkoxy, alkylthio, halides and O-containing heteroaryl groups. The N-containing heteroaryl group and the S-containing heteroaryl group may contain one or more further substituents, preferably selected from the group consisting of: amino, N-alkylamino, N,N-dialkylamino, thiol, hydroxy, alkyl, alkoxy, alkylthio, formyl, cyano, isocyanide, ethynyl, nitro, halide, hydroxycarbonyl, alkoxycarbonyl and O-containing heteroaryl groups, preferably alkyl, alkoxy, alkylthio, halides and O-containing heteroaryl groups. Examples of O-containing heteroaryl groups include, but are not limited to: furanyl, benzofuranyl, isobenzofuranyl, oxazolyl, isoxazolyl, benzoxazolyl, and oxadiazolyl. A preferred Raman active reporter compound is a compound having the general formula (I). in which A1, B1, and C1 are independently selected from N, CR16, and CR17, with the condition that only one of A1, B1, and C1 is N. A2, B2 and C2 are independently selected from N, CR18 and CR19, with the condition that only one of A2, B2 and C2 is N. E1, D1, E2, D2, R16, R17, R18 and R19 are independently selected from the following: hydrogen, amino, N-alkylamino, N,N-dialkylamino, thiol, hydroxy, alkyl, alkoxy, alkylthio, formyl, cyano, isocyanide, alkynyl, nitro, halide, hydroxycarbonyl, alkoxycarbonyl and heteroaryl groups containing O, preferably from hydrogen, alkyl, alkoxy, alkylthio, halides and heteroaryl groups containing O and X is a single bond or a linker -L2- selected from the following bonds: —CR8═CR9—, —N=N—, —≡—, —CR10═CR11— o -C6H4—, —CR10═CR11— m -C6H4—, —CR10═CR11— p -C6H4—, —CR10═CR11— o -C6H4—CR12═CR13—, —CR10═CR11— m -C6H4—CR12═CR13—, —CR10═CR11— p -C6H4—CR12═CR13—, —CR14═N—N═CR15—, , , , , , , , , , , , , , , , , , , , , , , , and ; wherein R8-R15 are selected from hydrogen, alkyl, alkoxy, alkylthio, formyl, cyano, nitro, halide, hydroxycarbonyl and alkoxycarbonyl. Preferably, the residues A1 and A2 in the general formula (I) are N. More preferably, the residues A1 and A2 are N and the substituents E1, D1, E2, D2, R16, R17, R18 and R19 in the general formula (I) are hydrogen. Raman-active reporter molecules without limitation include: 2-mercaptopyridine; benzenethiol; mercaptobenzoic acid; 4-nitrobenzenethiol; 3,4-dichlorobenzenethiol; 3-fluorothiophenol; 4-fluorothiophenol; 3,5-bis(trifluoromethyl)benzenethiol; 4-mercaptophenol; Biphenyl-4-thiol, 7- mercapto-4- methylcoumarin, 1-(4-hydroxyphenyl)-1H-tetrazol-5-thiol, 2-fluorothiophenol, 2-naphthalenethiol, 4-(((3-mercapto-5-(2-methoxyphenyl)-4H-2,1,4-triazol-4-yl)imino)methyl)phenol, (2-trifluoromethyl)benzenethiol, 4-aminothiophenol, 1-naphthalenethiol, 1,1',4,1"-terphenyl-4-thiol, biphenyl-,4,'4-dithiol, thiosalicylic acid, 4-(((3-mercapto-5-(2-pyridinyl)-H41-,2,4-triazol-4-yl)imino)methyl)-2,1-benzenediol, 4-(((3-mercapto-5-(2-pyridinyl)-H41-,2,4-triazol-4-yl)imino)methyl)benzoic acid, 2,3,4,6-tetrafluorobenzenethiol, (5-(4-methoxyphenyl)-1,3,4-oxidazole-2-thiol), (E) 1,2-di(pyridin-4-yl)ethene, 5-(pyridin-4-yl)-1,3,4-oxadiazole-2-thiol, and 4,1-bis((E)-2-(pyridin-4-yl)vinyl)benzene. The first colloid presented in step a) is characterized by a ζ potential value less than or equal to -25 mV. The second colloid presented in step b) is characterized by a ζ potential value less than or equal to -25 mV. As used herein, the ζ potential value of a colloid refers to the ζ potential value measured for said colloid at a concentration of 0.05 mg nanoparticle material per ml using a Malvern Zetasizer Nano-ZS with 1 ml double capillary cells. If necessary, for colloids with a concentration higher than 0.05 mg nanoparticle material per ml, the colloid is diluted with deionized water to a concentration of 0.05 mg nanoparticle material per ml before measuring the ζ potential. Preferably, the concentration of the nanoparticle material (mg / mL) in the first colloid and in the second colloid is less than 0.66 mg / mL, and more preferably between 0.05 mg / mL and about 0.30 mg / mL. For example, 0.05 mg / mL, 0.10 mg / mL, 0.15 mg / mL, 0.20 mg / mL, 0.25 mg / mL, and 0.30 mg / mL. After combining the first colloid with the second colloid, for example by simply adding the second colloid to the first colloid, the ratio between the number of nanoparticles of the first colloid and the number of nanoparticles of the second colloid is between about 1:25 to about 1:1, preferably from about 1:5 to about 1:1, preferably from about 1:4 to about 1:3, and causes the nanoparticles to aggregate. The aggregation described herein includes selective aggregation of the nanoparticles of the second colloid with the nanoparticles of the first colloid, i.e. there is no aggregation of the nanoparticles of the first colloid with the nanoparticles of the first colloid and there is no aggregation of the nanoparticles of the second colloid with the nanoparticles of the second colloid or if there is any aggregation, it is minor. Creating nanoparticle aggregation by any of the steps (D1-D3) or a combination of them: d1) The third colloid composition obtained in step c) with a pH between 2.2 and the lowest pH value at which the Raman active reporter molecules have a net electrical charge between 0 and 0.3. d2) Adding a salt solution, preferably a mineral salt solution, to the third colloid obtained in step c). d3) Adding a water-miscible solvent to the third colloid obtained in step c). In a preferred embodiment, inducing the aggregation of nanoparticles comprises step d1), namely mixing the third colloid obtained in step c) at a pH between 2.2 and the lowest pH value at which the Raman active reporter molecules have a net electrical charge between 0 and 0.3. The net electrical charge of a Raman active reporter molecule and the pH value corresponding to said net electrical charge can be determined via online tools such as Chemicalize.com (Chemicalize. ChemAxon. http: / / chemicalize.com / # / calculation) predicted that selective aggregation of the first colloidal nanoparticles with the second colloidal nanoparticles occurs at a pH between 2.2 and the lowest pH value where the Raman-active reporter molecules have a net electrical charge between 0 and 0.3. On the other hand, the induction of nanoparticle aggregation involves step d2), i.e. adding a salt solution, preferably a mineral salt solution, to the third colloid obtained in step c). Salt-related nanoparticle aggregation is a known method for inducing nanoparticle aggregation to those skilled in the art of colloidal chemistry (ChemPhysChem 2018, 19, 24-28). Examples of inorganic salts used as a solution in step d2) of the production method, such as an aqueous solution, include: sodium fluoride, sodium chloride, sodium bromide, sodium iodide, magnesium chloride, potassium chloride and mixtures thereof. In another alternative embodiment, the induction of nanoparticle aggregation comprises step d3), i.e. adding a water-miscible solvent to the third colloid obtained in step c). The water-miscible solvent is preferably selected from the list consisting of: methanol, ethanol, propanol, isopropanol, tetrahydrofuran, N-methyl-2-pyrrolidone (NMP), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), acetone and acetonitrile. A preferred production method according to the present invention comprises the following steps: a) Preparation of a first colloid consisting essentially of nanoparticles with a plasmonic surface that are approximately the same size and are dispersed in an aqueous solvent and a stabilizing agent is adsorbed on the surface of said nanoparticles and has a potential value ζ less than or equal to -25 mV; b) Preparation of a second colloid consisting essentially of surface plasmonic nanoparticles of approximately uniform size dispersed in an aqueous solvent. Raman active reporter molecules of general formula (I) and a stabilizing agent are adsorbed on the surface of said nanoparticles and have a potential ζ value less than or equal to -25 mV. c) To create the third colloid, the first colloid must be combined with the second colloid such that the ratio between the number of nanoparticles of the first colloid and the number of nanoparticles of the second colloid is between about 25:1 and 1:1, preferably from about 5:1 to 1:1, more preferably from 4:1 to about 3:1. d) Creating nanoparticle aggregation by any of the steps (d1-d3) or a combination of them: d1) mixing the third colloid obtained in step c) at a pH between 2.2 and about 6.1, preferably between 2.6 and about 5.7. d2) Adding a salt solution, preferably a mineral salt solution, to the third colloid obtained in step c). d3) adding a water-miscible solvent to the third colloid obtained in step c), and e) Stop the gathering. Preferably, step b) in the method claimed and described herein comprises the following steps, performed in order from b1) to b3): b1) preparing a colloid consisting of nanoparticles with a plasmonic surface that are approximately the same size and are dispersed in an aqueous solvent and a stabilizing agent is adsorbed on the surface of said nanoparticles and has a potential value ζ less than or equal to -25 mV; b2) adjusting the pH of the colloid to a value greater than the lowest pH value at which the Raman-active reporter molecules adsorbed on the surface of the nanoparticles have no net electrical charge, while the ζ potential value is less than or equal to -25 mV, preferably less than -40 mV, and b3) Adding a solution of Raman-active reporter molecules in a solvent to the colloid obtained in step b2), while the ζ potential value is less than or equal to -25 mV. The colloid provided in step b1) of the production method claimed herein essentially consists of nanoparticles having a plasmonic surface that are approximately the same size and are dispersed in an aqueous solvent and a stabilizing agent is adsorbed on the surface of said nanoparticles. The first colloid provided in step a) of the production method claimed herein essentially consists of nanoparticles having a plasmonic surface that are approximately the same size and are dispersed in an aqueous solvent and a stabilizing agent is adsorbed on the surface of said nanoparticles. The second colloid provided in step b) of the production method claimed herein essentially consists of nanoparticles having a plasmonic surface that are approximately the same size and are dispersed in an aqueous solvent, Raman active reporter molecules and a stabilizing agent are adsorbed on the surface of said nanoparticles. Therefore, the first colloidal nanoparticles and the second colloidal nanoparticles do not have molecules or organisms on their surface, which enables a specific interaction through a lock and key mechanism between the first colloidal nanoparticles and the second colloidal nanoparticles.Examples of these molecules include antibodies, proteins, antigens, complementary DNA strands and complementary RNA strands. Examples of these organisms include bacteria, viruses and spores. Therefore, the first colloid, the second colloid and also the colloid provided in step b1) are, in addition to being surfactant-free and polymer-free, free of molecules such as antibodies, proteins, antigens, complementary DNA strands and complementary RNA strands and organisms such as bacteria, viruses and spores. In step b2) of the production method according to the invention, the pH of the colloid is adjusted to a value above the lowest pH value at which the Raman active reporter molecules adsorbed on the surface of the nanoparticles have no net electrical charge. While the ζ potential value is less than or equal to -25 mV, preferably less than -40 mV. The net electrical charge of a Raman active reporter molecule and the pH value corresponding to said net electrical charge can be predicted by online tools such as Chemicalize.com (Chemicalize. ChemAxon. http: / / chemicalize.com / # / calculation). Preferably, in step b2, the pH of the colloid is adjusted to between 8.2 and about 12.1. While the ζ potential value is less than or equal to -25 mV, preferably less than -40 mV. In a more preferred embodiment, the pH of the colloid in step b2) is adjusted to a value of about 11.0. In step b3) according to the invention, a solution of the Raman active reporter molecule in a solvent is added to the colloid obtained in step b2). While the potential value ζ is less than or equal to -25 mV. The solvent used to prepare the solution of the Raman active reporter molecule includes any aqueous solvent and any organic solvent suitable for dissolving the Raman active reporter molecule. Examples of organic solvents include, but are not limited to, alcohols and are preferably selected from methanol, ethanol, propanol and isopropanol, tetrahydrofuran, N-methyl-2-pyrrolidone (NMP), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), acetone and acetonitrile. Aqueous solvents include, but are not limited to, water and mixtures of water and water-miscible solvents, such as methanol, ethanol, propanol, isopropanol, tetrahydrofuran, N-methyl-2-pyrrolidone (NMP), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), acetone, and acetonitrile. A preferred production method according to the present invention comprises the following steps: a) Preparation of a first colloid consisting essentially of nanoparticles with a plasmonic surface that are approximately the same size and are dispersed in an aqueous solvent and a stabilizing agent is adsorbed on the surface of said nanoparticles and has a potential value ζ less than or equal to -25 mV; b) Preparation of a second colloid consisting essentially of surface plasmonic nanoparticles of approximately uniform size dispersed in an aqueous solvent. Raman active reporter molecules of general formula (I) and a stabilizing agent are adsorbed on the surface of said nanoparticles and have a potential ζ value less than or equal to -25 mV. c) To create the third colloid, the first colloid must be combined with the second colloid such that the ratio between the number of nanoparticles of the first colloid and the number of nanoparticles of the second colloid is between about 25:1 and 1:1, preferably from about 5:1 to 1:1, more preferably from 4:1 to about 3:1. d) Creating nanoparticle aggregation by any of the steps (d1-d3) or a combination of them: d1) mixing the third colloid obtained in step c) at a pH between 2.2 and about 6.1, preferably between 2.6 and about 5.7. d2) Adding a salt solution, preferably a mineral salt solution, to the third colloid obtained in step c). d3) adding a water-miscible solvent to the third colloid obtained in step c) and e) Stop the gathering. Wherein, step b) includes the following steps, which are carried out in order from b1) to b3): b1) preparing a colloid consisting essentially of nanoparticles with a plasmonic surface that are approximately the same size and are dispersed in an aqueous solvent and a stabilizing agent is adsorbed on the surface of said nanoparticles and has a potential value ζ less than or equal to -25 mV; b2) adjusting the pH of the colloid to a value between 8.0 and about 12.1, preferably between 8.2 and 12.1, while the ζ potential value is less than or equal to -25 mV, preferably less than -40 mV, and b3) adding a solution of a Raman active reporter molecule of general formula (I) in a solvent to the colloid obtained in step b2), while the ζ potential value is less than or equal to -25 mV. The inventors have found that the signal intensity of the SERS tags produced according to the present invention can be enhanced by ensuring that a sub-monolayer or a layer of Raman-active reporter molecules is adsorbed onto the surface of the nanoparticles in the second colloid. Therefore, another preferred embodiment according to the present invention is directed to the method of producing SERS tags claimed herein, wherein the second colloid nanoparticles have a sub-monolayer or a layer of Raman-active reporter molecules adsorbed onto their surface. As used herein, a sub-monolayer of Raman-active reporter molecules refers to an incomplete monolayer of Raman-active reporter molecules. To ensure that a sub-monolayer or monolayer of Raman-active reporter molecules is adsorbed onto the surface of the second colloidal nanoparticles, the amount of Raman-active reporter molecules added in step b3) to the colloid obtained in step b2) is calculated by methods known to the skilled person, depending on the shape and size of the second colloidal nanoparticles. The production method claimed herein also enables the preparation of SERS tags consisting of a mixture of different Raman active reporter molecules, i.e. a mixture of two or more different Raman active reporter molecules. This is very advantageous, as it enables access to a variety of SERS tags, in which each of said SERS tags is characterized by a unique SERS signal, by combining a limited number of different Raman active reporter molecules in different ratios. To obtain such SERS tags, the second colloidal nanoparticles provided in step b) are prepared in such a way that they adsorb on their surface a mixture of different Raman active reporter molecules (i.e. a mixture of two or more different Raman active reporter molecules). Also, the second colloid can be prepared by using step b3) of the method of producing a solution of Raman active molecules in a solvent consisting of two or more different Raman active reporter molecules or by sequentially performing step b3) of this production method and using a different solution containing a Raman active reporter molecule each time.Therefore, another embodiment according to the invention is directed to a production method in which step b3) is a solution of Raman-active reporter molecules in a solvent comprising a mixture of two or more Raman-active reporter molecules. Another embodiment according to the invention is directed to a production method in which step b3) is carried out sequentially n times with n≥2, each time using a solution containing a Raman-active reporter molecule that is structurally different from the Raman-active reporter molecules used in the remaining n-1 solutions used in the remaining n-1 steps. As used herein, different Raman-active reporter molecules refer to Raman-active reporter molecules that have a different chemical structure and give a different SERS spectrum. To further increase the number of small particles and consequently the intensity of the SERS signal provided by the SERS tags produced by the production method claimed herein, it is preferred that in step c) the ratio between the number of first colloidal nanoparticles and the number of second colloidal nanoparticles is between 1:5 and about 1:1, preferably from about 1:4 to about 1:3. As shown, for example, in Example 10 and Figure 3b, the ratio between the number of first colloidal nanoparticles and the number of second colloidal nanoparticles is between about 5:1 and about 1:1, which allows access to SERS tags with a large number of small particles such as dimers, trimers and tetramers. To induce aggregation by the method described in step d1), it is necessary to mix the third colloid obtained in step c) with a pH between 2.2 and the lowest pH at which the Raman-active reporter molecules have a net electrical charge between 0 and 0.3, which for a Raman-active reporter molecule of general formula I) is between 2.2 and about 6.1, preferably between 2.6 and about 5.7. This can be achieved by adding an acid solution to the third colloid obtained in step c) or by adjusting the pH of the first colloid, so that the colloid obtained in step d1) has the required pH value. Accordingly, an embodiment according to the invention is directed to a production method wherein step d1) comprises adding an acid solution to the third colloid obtained in step c) during mixing, such that the pH of the resulting colloid is between 2.2 and the lowest pH value at which the Raman active reporter molecules have a net electrical charge of between 0 and 0.3, which in the case of the Raman active reporter molecule of general formula I) is between 2.2 and about 6.1, preferably between 2.6 and 5.7. Suitable acid solutions include, but are not limited to, acetic acid, hydrochloric acid and nitric acid. To avoid an additional production step, it is useful to adjust the pH of the first colloid, such that the colloid obtained in step d1) has a pH between 2.2 and the lowest pH value at which the Raman-active reporter molecules have a net electrical charge between 0 and 0.3, which in the case of a Raman-active reporter molecule of general formula (I), is between 2.2 and about 6.1, preferably between 2.6 and about 5.7. The production method claimed herein also comprises the step e) of stopping the aggregation. Preferably, step e) comprises any one of the following steps e1)-e4): E1) Adjusting the pH of the colloid obtained in step d) to a value higher than the lowest pH value at which the Raman-active reporter molecules adsorbed on the surface of the nanoparticles have no net electrical charge. E2) Diluting the colloid obtained in step d) with water, preferably so that the concentration of nanoparticles in the colloid is less than 6*109 nanoparticles per ml. e3) Adding a polymer to the colloid obtained in step d). e4) Adding a dielectric precursor to the colloid obtained in step d). In the inventive production method claimed herein, aggregation can be stopped by any of the methods e1)-e4). Adjusting the pH of the resulting colloid in step d) above the lowest pH value at which the Raman-active reporter molecules adsorbed on the surface of the nanoparticles have no net electric charge, as described in step e1), leads to an increase in the electrostatic repulsions between the nanoparticles, which in turn leads to the cessation of the aggregation process. The net electric charge of a Raman-active reporter molecule and the pH value corresponding to the said net electric charge can be determined through online tools such as Chemicalize.com (Chemicalize. ChemAxon. http: / / chemicalize.com / # / calculation) predicted. The addition of a polymer to the resulting colloid in step d) as described in step e3) results in an increase in the steric repulsion between the nanoparticles, which in turn leads to the cessation of the aggregation process. Advantageously, the polymer used in step e3) does not affect the SERS signal displayed by the SERS label. Suitable polymers include, but are not limited to, polyvinylpyrrolidone and polyethylene glycol. The addition of a dielectric precursor to the resulting colloid in step d) as described in step e4) results in the nanoparticle particles being encapsulated by at least one layer of dielectric material that stabilizes the SERS tags and implicitly stops the aggregation process. Preferably, the dielectric precursor is a silica precursor. Silica precursors include, but are not limited to, solutions of tetraethyl orthosilicate and 3-aminopropyl trimethoxysilane in ethanol, solutions of tetraethyl orthosilicate and (3-mercaptopropyl) trimethoxysilane in ethanol, solutions of sodium silicate in water, and solutions of (3-mercaptopropyl) trimethoxysilane in water. Alternatively, the aggregation process can be stopped by diluting the colloid obtained in step d) with water. Preferably, the aggregation is stopped by diluting the colloid obtained in step d) with water to reach a nanoparticle concentration [µg / mL] of less than or equal to 12.5 µg / mL (see e.g. E1 – E8, E11 – E14). Alternatively, the aggregation is stopped by diluting the colloid obtained in step d) with water to reach a nanoparticle concentration of less than 6 x 109 nanoparticles / mL. Another preferred embodiment of the invention is directed to a method of producing SERS tags, wherein step e) essentially comprises step e3) and the method of production further comprises diluting the colloid obtained in step e3) with water and / or coating the SERS tags with dielectric materials. In another preferred embodiment, steps c) and d) of the production method claimed herein are carried out simultaneously in a continuous flow system. In this particular production method, a continuous flow reactor is used. Such a continuous flow reactor is schematically shown in Fig. 1b and Fig. 1c and comprises two pressure vessels (111b, 112b, 111c, 112c) connected by a pipe to a tee (113b, 113c) and a collection vessel (114c, 114b). The second colloid is stored in the pressure vessel (111b, 111c), while the first colloid is stored in the pressure vessel (112b, 112c). A reservoir open to the atmosphere (114c, 114b) is used to collect the SERS tags and optionally to stop the aggregation (114c). A tube connecting each of the pressurized reservoirs (111b, 112b, 111c, 112c) to a tee (113b, 113c) is used to transfer the colloid. Another tube connecting the tee (113b, 113c) to a collection reservoir (114b, 114c) is used to perform the aggregation.The nanoparticles resulting from the aggregation are collected in the collection tank (114b), where the aggregation stops. When steps c) and d) of this production method are carried out simultaneously in a continuous flow system, it is preferred that step e) is also carried out in a continuous flow system. As mentioned above, the nanoparticles used in this method of producing SERS tags may have any shape such as a sphere, a pseudosphere, a rod, a disk, a prism, and a cube. Preferably, the shape of the surface plasmonic nanoparticles used in the inventive production method claimed herein is selected from a sphere and a pseudosphere. Even more preferably, said nanoparticles have a pseudospherical shape. In a preferred embodiment, the first colloid nanoparticles and the second colloid nanoparticles are of the same size. Conveniently, for such a method of producing SERS tags, the colloid used to prepare the second colloid (i.e., the colloid provided in step b1)) is the first colloid. In an alternative embodiment, the size of the nanoparticles of the first colloid is different from the size of the nanoparticles of the second colloid. For example, the size of the nanoparticles in the first colloid may be smaller than the size of the nanoparticles in the second colloid, resulting in the SERS tags having a structure similar to the SERS tags shown in Figure 3A, or the size of the nanoparticles in the first colloid may be larger than the size of the nanoparticles in the second colloid, resulting in the SERS tags having a structure similar to the SERS tags shown in Figure 3B. The production method claimed herein is preferably carried out with colloids in which the plasmonic surface of the nanoparticles in the first colloid and / or the plasmonic surface of the nanoparticles in the second colloid are made of gold, preferably with colloids containing solid gold nanoparticles and even more preferably with citrate-stabilized gold colloids. Examples The invention will now be explained in more detail with reference to the following non-limiting examples. General The following reagents were obtained from the following suppliers: Gold Chlorovate Trihydrate (>99.9%; Registry No.: 16961-25-4), Sodium Borohydride (99.99%; Registry No.: 2-66-16940), Sodium Citrate Tribasic Dihydrate (≥99.5%; Registry No.: 6132-04-3), (E)-1,2-Di(Pyridin-4-yl) Ethene (97%; Registry No.: (13362-78-2), 5-(Pyridin-4-yl)-4,3,1-Oxadiazole-2-thiol (97%; Registry No.: 15264-63-8), Sodium Hydroxide (ACS Reagent, ≥97%; Registry No.: 1310-73-2), Hydroxylamine Hydrochloride (99.999%); Registry No.: 5470-11-1), Terephthalaldehyde (Reagent Plus, 99%, Registry No.: (623-27-8), 4-methylpyridine (99%; Registry No.: 108-89-4), acetic anhydride (Reagent Plus, ≥99%; Registry No.: 108-24-7), dichloromethane (anhydrous, 99.8%; Registry No.: 75-09-2), and methanol (HPLC, ≥99.9%; Registry No.: 67-56-1) were purchased from Sigma Aldrich. Hydrochloric acid (rare metal grade, 34-37%; Registry No.: 7647-01-0) was purchased from Fisher Scientific. The ζ potential values ​​were measured using a Malvern Zetasizer Nano-ZS with 1 ml double capillary cells (DTS1060). The optical absorption spectra were recorded on an Agilent 8453 spectrophotometer and a Perkin Elmer Lambda 650. Scanning electron microscope (SEM) images were taken on a Hitachi S-4500. The 785 nm Raman spectrum was acquired on an Ocean Optics QE 6500. Colloidal gold particles of nominal 140 nm were measured by sending a sample to the EAG laboratory for transmission electron microscopy (TEM) imaging. Images were analyzed with ImageJ software (https: / / imagej.nih.gov / ij / ). A batch was considered nominally 140 nm if the average particle size was within ±9 nm of the nominal diameter. The average particle size was determined by measuring 230 individual particles in TEM micrographs. The size-related characteristic chosen to describe individual particles was the “circle equivalent” (CE) diameter, which is the diameter of a circle that has an area equal to the orthographic prominence of the particle. The diameter of gold nanoparticles d (nm) for 40, 60, and 90 nm nanoparticle assemblies was calculated by the method of Haiss et al. (Anal. Chem. 2007, 79, 4215-4221) using this equation. where λspr is the position of the surface plasmon resonance peak in the extinction diagram taken from the colloidal sample on a Perkin Elmer Lambda 650 UV Vis. A cluster was considered nominally 40 nm, 60 nm, or 90 nm if the position of the surface plasmon resonance peak corresponded to a diameter within ±9 nm of the nominal diameter. I. Preparation of colloidal gold storage solution (S1 – S4). Gold colloidal stock solutions (S1 - S4), characterized by gold nanoparticle size (nm), gold concentration (mg / mL), and pH values ​​shown in Table 1, were produced as follows: I.1 Preparation of 40 nm gold colloidal stock solution (S1) In a clean 100-liter glass reactor (ChemGlass), 79.5 liters of 17 MΩ water were cooled to 0.5±3.5°C. While stirring with a paddle wheel at 400 rpm, gold chlorovate trihydrate (100 g) was added as a 20 wt% solution in water. Sodium citrate tribasic dihydrate (.3174 g) as a 30 wt% solution in water and hydroxylamine hydrochloride (6.155 g) as a 23.5 wt% solution in water were combined and added to the reactor. After 10 seconds, 800 μL of sodium borohydride as a 0.063 wt% solution in 0.01 N sodium hydroxide was injected into the reactor. The reactants were allowed to react for 2 min and then discharged into a clean drum to prepare a 40 nm colloidal gold stock solution (S1) with a gold concentration of 0.25 mg gold / mL and a pH value of approximately 2.4. I.2 Preparation of 60 nm gold colloidal stock solution (S2) In a clean 100-liter glass reactor (ChemGlass), 79.5 liters of 17 MΩ water were cooled to 0.5±3.5°C. While stirring with a paddle wheel at 400 rpm, gold chloroaluminum trihydrate (200 g) was added as a 20 wt% solution in water. Sodium citrate tribasic dihydrate (173.3 g) as a 30 wt% solution in water and hydroxylamine hydrochloride (217.1 g) as a 17.5 wt% solution in water were combined and added to the reactor. After 10 seconds, 800 μL of sodium borohydride as a 0.052 wt% solution in 0.01 N sodium hydroxide was injected into the reactor. The reactants were allowed to react for 2 minutes and then discharged into a clean drum. This batch was diluted to 160 L with 17 MΩ water to obtain a 60 nm gold colloidal stock solution (S2) with a gold concentration of 0.25 mg gold / mL and a pH of approximately 2.4. I.3 Preparation of 90 nm gold colloidal stock solution (S3) In a clean 100 L glass reactor (ChemGlass), 79.5 L of 17 MΩ water was cooled to 0.5 ± 3.5 °C. While stirring with a paddle wheel at 400 rpm, gold chlorovate trihydrate (200 g) was added as a 20 wt% solution in water. Sodium citrate tribasic dihydrate (3.173 g) as a 30 wt% solution in water and hydroxylamine hydrochloride (1.217 g) as a 17.5 wt% solution in water were combined and added to the reactor. After 10 s, 900 μL of sodium borohydride as a 0.01 wt% solution in 0.01 N sodium hydroxide was injected into the reactor. The reactants were allowed to react for 2 min and then discharged into a clean drum. This batch was diluted to 160 L with 17 MΩ water to obtain a 90 nm gold colloidal stock solution (S3) with a gold concentration of 0.25 mg gold / mL and a pH of approximately 2.4. I.4 Preparation of 140 nm gold colloidal stock solution (S4) In a 2-liter glass vessel, 1.5 liters of 17 MΩ water were stirred at room temperature. Gold chlorovate trihydrate (2.5 g) was added as a 20 wt% solution in water while stirring. Sodium citrate tribasic dihydrate (15.37 g) as a 30 wt% solution in water and hydroxylamine hydrochloride (8.37 g) as a 16.7 wt% solution in water were combined and added to the reactor. After 5 seconds, 25 μL of sodium borohydride as a 0.01 wt% solution in 0.01 N sodium hydroxide was injected into the reactor. Additional stirring for 15 minutes provided a 140 nm gold colloidal solution (S4) with a gold concentration of 0.25 mg gold / mL and a pH of approximately 2.4. Table 1: Characteristics of colloidal gold storage solutions (S1 – S4). Gold colloidal stock solution number Gold nanoparticle size (nm) Gold concentration (mg / mL) pH S1 40 0.25  2.4 S2 60 0.25  2.4 S3 90 0.25  2.4 S4 140 0.25  2.4 #x200eII #x200e#x200f. #x200fPreparation of the first colloid (A1 – A9) (step A) with this production method The first colloid (A1 – A9) was obtained starting from the gold colloidal solutions S1, S2 and S4. If necessary (e.g.: gold colloids A1 and A7) the gold colloidal stock solutions were diluted with deionized water to obtain the gold concentration (mg / mL) shown in Table 2. Furthermore, if necessary (e.g.: gold colloids A3, A4, A5, A6, A8, A9), the gold colloidal stock solutions were considered either on their own or after dilution with a 0.1 mM aqueous solution of NaOH or a 0.1 mM aqueous solution of HCl to bring the pH value of the first colloids to the pH value shown in Table 2. Table 2: Properties of the first colloids (A1 – A9). Gold colloidal stock solution number Gold nanoparticle size (nM) Gold concentration (mg / mL) pH value A1 60 0.10 2.5 A2 60 0.25 2.4 A3 60 0.25 3.0 A4 60 0.25 1.7 A5 60 0.25 8.8 A6 60 0.25 4.2 A7 140 0.10 2.4 A8 40 0.25 4.2 A9 60 0.25 6.0 Only stable initial colloids, i.e. colloids in which there is no aggregation, are suitable for use in the production method according to the invention. The initial colloids A1 - A9 are stable, i.e. no aggregation of gold nanoparticles can be detected by visual detection of a color change from pink to purple. Furthermore, as shown in Figure 4, the types of gold colloids measured with a zeta potential value less than or equal to -25 mV are stable, as described in Case IV below. Therefore, according to the invention, such gold colloids are also useful as initial colloids in this production method. #x200eIII#x200e#x200f. #x200fPreparation of the second colloid containing gold nanoparticles and Raman-active reporter molecules adsorbed on the surface of gold nanoparticles (D1 – D11) (step B) by this production method Gold colloids (B1–B8), characterized by the gold concentration (mg / mL) and pH values ​​shown in Table 3, were prepared starting from gold colloid stock solutions S1–S3. If necessary, the preparation included (e.g. gold colloids B1 and B5) gold colloidal solutions diluted with deionized water to obtain the specified gold concentration (mg / mL) and the pH value was adjusted by adding a 1 M aqueous solution of NaOH to the gold colloidal stock solutions either on their own or after dilution. Table 3: Properties of gold colloids B1 – B9. Gold colloid number Gold nanoparticle size (nM) Gold concentration (mg / mL) pH value B1 60 0.10 11.2 B2 60 0.25 11.2 B3 60 0.25 9.8 B4 60 0.25 12.1 B5 90 0.10 11.0 B6 60 0.25 7.1 B7 60 0.25 5.1 B8 40 0.25 11.0 B9 60 0.25 8.2 The Raman-active reporter molecule 4,1-bis((E)-2-(pyridin-4-yl)vinyl)benzene was synthesized as follows: In a 50 mL round-bottom flask with a stir bar, terephthalaldehyde (3.44 g, 25.7 mmol), 4-methylpyridine (9.57 g, 103 mmol), and acetic anhydride (25 mL) were charged. The mixture was stirred until no more aldehyde was present (4 h, as monitored by TLC). The reaction was cooled to room temperature and quenched by pouring into 100 mL of ice water. The cooled mixture was neutralized to pH 7 using 6 N aqueous NaOH, and the resulting brown precipitate was filtered, washed with water, and air-dried. Extraction with dichloromethane followed by concentration of the solvent to dryness provided a crude product that was purified by flash column chromatography on silica gel (methanol / dichloromethane: 95.5) to afford 0.875 g of the target Raman-active reporter molecule as a yellow solid (12%). The second colloid (D1-D11) containing gold nanoparticles adsorbing Raman-active reporter molecules on their surface was prepared by adding a volume of 0.1 mM solution of Raman-active reporter molecule in ethanol to 20 mL of gold colloid (B1-B5, B9) or L3 gold colloid (B8), and then the resulting mixture was stirred for 30 min (second colloid D1-D7, D9-D11) / 1 h (second colloid D8) at room temperature. Table 4 provides a summary of the Raman-active reporter molecules and the volume of Raman-active reporter molecule solution in ethanol used to prepare the second colloid D1-D11. Table 4: Preparation of the second colloid D1 – D11. Colloid number II Start of gold colloid Raman active reporter molecule Volume 0.1 mM Solvent Raman active reporter molecule D1 B1 1-(E),2-di(pyridin-4-yl)ethene 300 µL D2 B2 -(E),2-di(pyridin-4-yl)ethene 750 µL D3 B3 -(E),2-di(pyridin-4-yl)ethene 750 µL D4 B4 -(E),2-di(pyridin-4-yl)ethene 750 µL D5 B2 5-(pyridin-4-yl)-4,3,1-oxadiazole-2-thiol 750 µL D6 B2 4,1-bis((E)-2-(pyridin-4-yl)vinyl)benzene 750 µL D7 B5 -(E),2-di(pyridin-4-yl)ethene 240 µL D8 B8 -(E),2-di(pyridin-4-yl)ethene 127.5 mL D9 B9 -(E),2-di(pyridin-4-yl)ethene 750 µL D10 B1 4,1-bis((E)-2-(pyridin-4-yl)vinyl)benzene 300 µL D11 B1 5-(pyridin-4-yl)-4,3,1-oxadiazole-2-thiol 300 µL Yield 20 mL of gold colloids B6 and B7 having a pH value below the lowest pH value at which 1-(E),2-di(pyridin-4-yl)ethene has no net electrical charge. As predicted by Chemicalize.com, 750 μL of a 0.1 mM solution of 1-(E),2-di(pyridin-4-yl)ethene in ethanol followed by stirring the resulting mixture for 30 minutes at room temperature resulted in the formation of unstable colloids, as determined by visual detection of a color change from pink to purple of the mixture upon stirring. According to the invention, unstable colloids cannot be used in this production method. To ensure the stability of the second colloid, i.e., to prevent the aggregation of gold nanoparticles in the colloid, it is important that during the preparation and storage of the colloid, the ζ potential value is less than or equal to -25 mV and the pH value is higher than the lowest pH value at which the Raman-active reporter molecule that will be adsorbed on the gold nanoparticles has no net electrical charge, which can be predicted, for example, with Chemicalize.com.This means that the pH of the gold colloid (B1 - B5, B8, B9) used to prepare the second colloid is adjusted to a higher value than the lowest pH before activation with the Raman-active reporter molecule. The Raman-active reporter molecule adsorbed on the gold nanoparticles in the gold colloid (B1 - B5, B8, B9) has no net electric charge, which can be predicted, for example, with Chemicalize.com. IV. Stability of the first and second colloids. In the production method according to the invention, for proper use and provision of SERS tags with a large number of small particles, the first and second colloids must be stable, i.e., aggregation of nanoparticles must be avoided during preparation and storage of the colloids. This can be ensured by maintaining the ζ potential value of said colloids at a value less than or equal to -25 mV. As shown in Figure 4 and Table 5, various gold colloids can be used as the first and second colloids in the production method of SERS tags according to the invention. The ζ potential of the first colloid A2 and the second colloids D2, D5 and D6 were measured as a function of pH at a concentration of 0.05 mg gold / mL at room temperature. The results are reported in Table 5 and shown in Figure 4. ζ potential measurements were performed on a Malvern Zetasizer Nano-ZS with 1 mL double capillary cells. Physical properties of water at 25 °C and gold preloaded onto the device. The first colloid A2 and the second colloids D2, D5 and D6 were each diluted with deionized water to a concentration of 0.05 mg gold / mL. The ζ potential values ​​of the first colloid A2 and the second colloids D2, D5 and D6 at the pH range used to synthesize the SERS tags according to the invention are reported in the second column of Table 5. The ζ potential values ​​of the first colloid A2 and the second colloids D2, D5 and D6 at the pH at which the colloids become unstable are reported in the third column of Table 5. Colloid instability was determined by visual detection of a change in colloid color from pink to purple. Table 5: Measurement of the ζ potential of the first and second colloids. Gold colloid number diluted at 0.05 mg Au / mL [Gold concentration] ζ potential after dilution (pH after dilution) ζ potential at which the gold colloid becomes unstable (pH at which the gold colloid becomes unstable) A2 -43 mV (at pH  2.5) -17 mV (at pH  1.6) D2 -44 mV (at pH  10) -22 mV (at pH  6) D5 -38 mV (at pH  10) -16 mV (at pH  3.4) D6 -36 mV (at pH  10) -23 mV (at pH  7) V. Combining the first colloid and the second colloid, inducing aggregation and stopping aggregation (steps c), d) and e) of this production method) 1.V SERS tags containing gold nanoparticles of the same size (e.g. E1 - E8, E12 - E14) 1.V.A. Batch aggregation (Examples E1 - E7, E12 - E14) The second colloid (D1–D6, D9–D11), 20 mL, was quickly poured into 80 mL of the first colloid (A1–A5, A9) and the mixture was stirred in a mixing vessel with a magnetic stir bar. 30 s after the colloids were combined, a 125 μL aliquot was withdrawn and diluted to 1 mL with water, thereby stopping aggregation. At this dilution (12.5 μg gold / mL corresponding to approximately 5.72 × 109 gold nanoparticles / mL), aggregation was stopped and the SERS signal was measured using 785 nm laser excitation and a QE65000 spectrometer purchased from Ocean Optics Inc. with an integration time setting of one second. The results of the SERS signal measurements performed on the fabricated SERS tags, as described above, are shown in Table 6. As confirmed by Examples E1-E7 and E12-E14 according to the inventive method claimed herein and Comparative Examples C1-C3 and as predicted by Chemicalize.com, selective aggregation of the first colloid particles and the second colloid particles does not occur at low pH values ​​such as 1.8 and at pH values ​​above the lowest pH value at which the Raman active reporter molecules have a net electrical charge between 0 and 0.3. Table 6: SERS signal intensity indicated by SERS labels corresponding to Examples E1–E7, E12 and Comparative Examples C1–C3. Example No. First colloid No. Second colloid Ratio between the number of nanoparticles of the first colloid and the number of nanoparticles of the second colloid pH Colloid obtained by the mixture of the first and second colloids SERS signal intensity 12.5 in μg Au / mL [cps ± 500] E1 A1 D1  4:1  2.9 9385 E2 A2 D2  4:1  2.6 14321 E3 A3 D2  4:1  3.2 17091 E4 A2 D3  4:1  3.3 7248 E5 A3 D4  4:1  5.7 14598 E12 A2 D9  4:1  2.6 11109 C1 A4 D2  4:1  1.8 3270 (no aggregation) C2 A9 D2  4:1  6.5 512 (without accumulation) C3 A5 D2  4:1  9.9 373 (without accumulation) E6 A2 D5  4:1  2.6 15617 E13 A1 D11  4:1  2.9 13654 E7 A2 D6  4:1  2.6 28993 E14 A1 D10  4:1  2.9 24196 1.V.B. Continuous flow system accumulation (Example E8) A schematic of the continuous flow reactor used in the production method according to the invention is presented in Figure 1b. A pressure vessel (111b) holds the second colloid and a second pressure vessel (112b) holds the first colloid. A vented vessel (114b) is used to collect the SERS tags and contains a concentrated polymer solution to stop the aggregation reaction. Transparent fluorinated ethylene propylene (FEP) tubing from Cole Parmer®Scientific Experts with a nominal internal diameter of 8 mm is used to transport the colloid. A 30 cm length of FEP tubing was used to connect the pressure vessel (111b) to the tee fitting (113b). A 30 cm length of FEP tubing was used to connect the pressure vessel (112b) to the tee fitting (113b). 7 meters of FEP tubing were used to connect the tee (113b) to the collection tank (114b). This section of tubing between the tee (113b) and the collection tank (114b) is where the accumulation occurs. The colloids A8 (12 L) and D8 (3 L) described above were placed in tanks 112b and 111b, respectively. Tank (111b) containing the second colloid (D8) was pressurized such that the colloidal flow rate was 0.66 L / min. Tank (112b) containing the first colloid (A8) was pressurized such that the colloidal flow rate was 2.65 L / min. After mixing at the T-junction, the combined colloid, characterized by a ratio of 4:1 between the number of nanoparticles of the first colloid A8 and the number of nanoparticles of the second colloid D8, had a pH value of about 4.8 and a flow rate in the collection tube of 3.31 L / min, giving a linear velocity of 1.1 m / s. SERS measurements were performed through a transparent FEP tube at a 1-m mark along the length of the collection tube with a probe coupled to a fiber optic cable to an Ocean Optics QE65000 spectrometer with 785 nm excitation. The integration time was shortened such that the measurement at the mark at the end of the tube was equal to the value obtained from a 50 μl aliquot sampled at the mouth of the tube and diluted to 1 ml with water (12.5 μg gold / ml), i.e., to a sample in which aggregation was stopped by dilution with water, and measured using 785 nm laser excitation and an Ocean Optics QE65000 spectrometer with an integration time of one second. The results of the SERS measurements are plotted in Figure 5 as diamonds connected by a solid line. The SERS signal intensity from the sample taken at the end of the tube and measured at 12.5 μg gold / ml was 11,044 counts / s [±500]. 2.V The first colloid and the second colloid contain gold nanoparticles of different sizes (Examples E9 - E11) 2.V.A. Batch aggregation (Example E9 and E10) Example E9 The second colloid (D7, 30 mL) was quickly poured into the first colloid (A1, 55 mL) and the mixture was stirred in the mixing tank with a magnetic stir bar. The ratio between the number of nanoparticles of the first colloid and the number of nanoparticles of the second colloid was 6.3:1. After 30 s of mixing at a pH of approximately 2.9, aggregation was stopped by adding a polymer solution and the resulting particles were coated with silica to provide target SERS labels as described in US8497131B2. ~2 μL of SERS labels were dropped onto silica wafers and dried. The sample was imaged on a Hitachi S-4500 field emission SEM and is shown in Fig. 3a. The SERS signal was measured using 785 nm laser excitation and a QE65000 spectrometer purchased from Ocean Optics, set to an integration time of one second. The measured SERS signal intensity at 12.5 μg gold / mL was 18,377 counts / s [±500]. Example E10 The second colloid (D7, 20 mL) was quickly poured into the first colloid (A7, 200 mL) and the mixture was stirred in the mixing tank with a magnetic stir bar. The ratio between the number of nanoparticles of the first colloid and the number of nanoparticles of the second colloid is 2.65:1. After 30 seconds of mixing at pH approximately 2.9, aggregation was stopped by the addition of polymer solution and the resulting particles were coated with silica to provide target SERS labels as described in US8497131B2. ~2 μL of SERS labels were dropped onto silica wafers and dried. The sample was imaged on a Hitachi S-4500 field emission SEM and is shown in Figure 3b. 2.V.B. Accumulation of flow system (Example E11) A schematic of the continuous flow reactor used in the production method according to the invention is presented in Figure 1b. A pressure vessel (111b) holds the second colloid and a second pressure vessel (112b) holds the first colloid. A vessel open to the atmosphere (collection vessel, 114b) is used to collect the SERS tags and stop the aggregation. Fluorinated ethylene propylene (FEP) tubing from Cole Parmer®Scientific Experts with a nominal inner diameter of 8 mm is used to transport the colloid. A 30 cm length of clear FEP tubing was used to connect the pressure vessel (111b) to the tee fitting (113b). A 30 cm length of FEP tubing was used to connect the pressure vessel (112b) to the tee fitting (113b). A 7 m length of FEP tubing was used to connect the tee fitting (113b) to the collection vessel (114b). This section of pipe between the tee connection (113b) and the collection tank (114b) is where the accumulation occurs. The A6 (12 L) and D8 (3 L) colloids described above are placed in tanks 112b and 111b, respectively. Tank (111b) containing the second colloid (D8) was pressurized such that the colloidal flow rate was 0.66 L / min. Tank (112b) containing the first colloid (A6) was pressurized such that the activated colloid flow rate was 2.65 L / min. After mixing at the T-junction, the combined colloid, characterized by a ratio of 1:1.2 between the number of nanoparticles of the first colloid A6 and the number of nanoparticles of the second colloid D8, had a pH of about 4.8 and a flow rate in the collection tube of 3.31 L / min, giving a linear velocity of 1.1 m / s. SERS measurements were performed through a transparent FEP tube at a 1-m mark in the length of the collection tube with a probe coupled to a fiber optic cable to an Ocean Optics QE65000 spectrometer with excitation at 785 nm. The integration time was shortened such that the measurement at the mark at the end of the tube was equal to the value obtained from a 50 μl aliquot sampled at the mouth of the tube and diluted to 1 ml with water (12.5 μg gold / mL) was diluted (step e2) and measured using 785 nm laser excitation and an Ocean Optics QE65000 spectrometer with an integration time of one second. The results of these measurements are plotted in Figure 5 as circles connected by a dotted line. The SERS signal intensity from the sample taken at the end of the tube and measured at 12.5 μg gold / mL was 28,303 counts / s [±500].

Claims

Amended Claim 1. A method for producing surface-enhanced Raman spectroscopy (SERS) tags comprising the steps of: a) providing a first colloid consisting essentially of surface-plasmonic nanoparticles that are approximately the same size and are dispersed in an aqueous solvent, and a stabilizing agent adsorbed on the surface of said nanoparticles and having a ζ potential value less than or equal to -25 mV; b) providing a second colloid consisting essentially of surface-plasmonic nanoparticles that are approximately the same size and are dispersed in an aqueous solvent. Raman-active reporter molecules and a stabilizing agent adsorbed on the surface of said nanoparticles and having a ζ potential value less than or equal to -25 mV. c) To create the third colloid, the first colloid must be combined with the second colloid such that the ratio between the number of nanoparticles of the first colloid and the number of nanoparticles of the second colloid is between 1:25 and 1:1, d) creating the aggregation of nanoparticles by any of the steps (d1-d3) or a combination of them: d1) combining the third colloid obtained in step c) with a pH between 2.2 and the lowest pH value at which the Raman active reporter molecules have a net electric charge between 0 and 0.

3. d2) adding a salt solution to the third colloid obtained in step c). d3) adding a water-miscible solvent to the third colloid obtained in step c) and e) stopping the aggregation.

2. The method according to claim 1, step b) comprising the following steps, which are carried out in order from b1) to b3): b1) preparing a colloid consisting of nanoparticles with a plasmonic surface that are approximately the same size and are dispersed in an aqueous solvent and a stabilizing agent is adsorbed on the surface of said nanoparticles and has a ζ potential value less than or equal to -25 mV; b2) adjusting the pH of the colloid to a value higher than the lowest pH value at which the Raman-active reporter molecules adsorbed on the surface of the nanoparticles have no net electrical charge. While, the ζ potential value is less than or equal to -25 mV, and b3) adding a solution of the Raman-active reporter molecules in a solvent to the colloid obtained in step b2), while the ζ potential value is less than or equal to -25 mV.

3. The method according to claim 1 or 2, wherein the stabilizing agent is selected from carboxylic acids, carboxylic acid salts, phosphoric acids, phosphoric acid salts, ascorbic acid, ascorbic acid salts, and mixtures thereof.

4. The method according to any one of claims 1 to 3, wherein the second colloidal nanoparticles have adsorbed on their surface a submonolayer or monolayer of Raman-active reporter molecules.

5. The method according to any one of claims 1 to 4, wherein in step b3) the solution of Raman active reporter molecules in a solvent comprises a mixture of two or more different Raman active reporter molecules.

6. The method according to any one of claims 1 to 5, wherein in step c) the ratio between the number of first colloidal nanoparticles and the number of second colloidal nanoparticles is between 1:4 and 1:

3.

7. The method according to any one of claims 1 to 6, wherein step d1) comprises adding an acid solution to the third colloid obtained in step c) during mixing, such that the pH value of the resulting colloid is between 2.2 and the lowest pH value at which the Raman active reporter molecules have a net electrical charge between 0 and 0.

3.

8. The method according to any one of claims 1 to 6, wherein the pH of the first colloid is adjusted such that the pH of the third colloid obtained in step d1) is between 2.2 and the lowest pH value at which the Raman active reporter molecules have a net electrical charge between 0 and 0.

3.

9. The method according to any one of claims 1 to 8, wherein step e) comprises any of the following steps from e1)-e4): e1) adjusting the pH of the colloid obtained in step d) to a value higher than the lowest pH value at which the Raman-active reporter molecules adsorbed on the surface of the nanoparticles have no net electrical charge. e2) diluting the colloid obtained in step d) with water; e3) adding a polymer to the colloid obtained in step d). e4) adding a dielectric precursor to the colloid obtained in step d).

10. The method according to any one of claims 1 to 9, wherein step e) essentially comprises step e3), and the production method further comprises diluting the colloid obtained in step e3) with water and / or coating the SERS tags with a dielectric material.

11. The method according to any one of claims 1 to 10, wherein steps c) and d) are carried out simultaneously in a continuous flow system.

12. The method according to claim 11, wherein step e) is carried out in a continuous flow system.

13. The method according to any one of claims 1 to 12, wherein the first colloidal nanoparticles and the second colloidal nanoparticles have the same size.

14. The method according to any one of claims 1 to 12, wherein the size of the first colloidal nanoparticles is different from the size of the second colloidal nanoparticles.

15. The method according to any one of claims 1 to 14, wherein the plasmonic surface of the nanoparticles in the first colloid and / or the plasmonic surface of the nanoparticles in the second colloid are made of gold.

Images