RU2804303C2 - Method for manufacturing surface-enhanced raman spectroscopy tags - Google Patents

Abstract

FIELD: SERS tags.

SUBSTANCE: method for manufacturing surface-enhanced Raman spectroscopy (SERS) tags involves the following steps: a) obtaining the first colloid, actually consisting of nanoparticles with a plasmonic surface and essentially the same size, dispersed in an aqueous solvent, and a stabilizing agent adsorbed on the surface of these nanoparticles and characterized by the value ξ - potential less than or equal to -25 mV; b) obtaining the second colloid, actually consisting of nanoparticles with a plasmonic surface and essentially the same size, dispersed in an aqueous solvent, active Raman scattering reporter molecules adsorbed on the surface of the said nanoparticles, and a stabilizing agent adsorbed on the surface of the said nanoparticles, and characterized by the value ξ - potential less than or equal to -25 mV; c) combining the first colloid with the second colloid so that the ratio of the first colloid nanoparticles to the second colloid nanoparticles is from about 25:1 to about 1:1, preferably from about 5:1 to about 1:1 to form the third colloid; and d) inducing aggregation of the nanoparticles. In this case, induction of aggregation of nanoparticles occurs using any of the steps d1)-d3) or a combination thereof: d1) mixing the third colloid obtained in step c) at a pH in the range from approximately 2.2 to the lowest pH value at which the active reporter molecules of Raman scattering have a total electric charge from 0 to 0.3; d2) adding a salt solution, preferably an inorganic salt solution, to the third colloid obtained in step c); d3) adding a water-soluble solvent to the third colloid obtained in step c); and e) stopping aggregation.

EFFECT: ability to expediently produce large quantities of SERS-tags, characterized by a narrow size distribution and a high proportion of aggregates with a small number, SERS-tags provide increased SERS responses in the group.

15 cl, 5 dwg, 6 tbl

Description

Field of technology to which the invention relates

The present invention relates to the field of methods for making surface enhanced Raman spectroscopy (SERS) tags. The production method according to the present invention is reproducible and versatile and allows the expedient production of large quantities of SERS tags characterized by a narrow size distribution and a high proportion of low-number aggregates. SERS tags manufactured by the claimed manufacturing method described herein provide enhanced SERS responses in a group.

Prerequisites for creating the invention

Surface enhanced Raman spectroscopy (SERS) tags have proven useful for a variety of applications, including product labeling for identification and authentication purposes, and high-throughput multiplex screening in microarray technology, diagnostics, and bioimaging. SERS tags are aggregates of nanoparticles with a plasmonic surface and active Raman reporter molecules adsorbed on their surface. The plasmonic surface nanoparticles are responsible for generating the electric field required for Raman enhancement, while the active Raman reporter molecule provides the unique vibrational fingerprint of the SERS tag. Typically, the aggregates are an outer coating layer that a) isolates the SERS tag from the external environment, thereby preventing the leaching of active Raman reporter molecules from the SERS tag and protecting the SERS tag from environmental contamination that can cause vibration noise, b ) increases the colloidal stability of the SERS tag and c) provides a convenient surface for further chemical functionalization. Today, polymers and silicon dioxide are used as outer coating layers.

Due to the strong dependence of plasmonic properties on the aggregated states of nanoparticles, the production of SERS tags with a large number of low-number aggregates is highly desirable to obtain enhanced SERS responses in a group. SERS tags with a large number of aggregates with a small number were obtained either using methods postsynthetic sorting, or using controlled aggregation synthesis processes.

For example, field-flow fractionation applied to a mixture containing individual nanoparticles and nanoparticle aggregates ranging in size from dimers to octamers resulted in a fraction enriched in dimeric (10%), trimeric (21%), and tetramic (13%) SERS- labeled, but which nevertheless contained a high percentage of single nanoparticles (52%) ( J. Am. Chem. Soc. 2010, 132 , 10903-10910). A mixture containing individual nanoparticles and aggregates ranging in size from dimers to dodecamers was enriched with dimeric (52%) and trimeric (32%) SERS tags using a post-synthetic centrifugal sorting method in a high-viscosity density gradient medium, such as an aqueous iodixanol density gradient medium. (US9802818B2). In addition to the fact that post-synthetic sorting methods are labor-intensive and resource-intensive, they require the use of harsh conditions (in the case of, for example, highly viscous reagents), which lead to destabilization of uncoated SERS tags. Thus, post-synthetic sorting methods are only compatible with silica- or polymer-coated SERS tags. Therefore, aggregation-controlled synthesis processes are of great advantage for the production of SERS tags with a narrow size distribution and a high ratio of low-number aggregates.

Aggregation using a solid support made it possible to obtain SERS tags with a narrow size distribution. In Ruan's publication et al. (Adv. Optical Mater. 2014, 2, 65 - 73) describes the synthesis of SERS tags with asymmetric central and peripheral structures, having a certain number, i.e. 18 ± 2, peripheral structures in the form of Au nanospheres with an average diameter of 24 nm per central structure in the form of an Au nanosphere with an average diameter of 180 nm. The synthesis involves the adsorption of cetyltrimethylammonium bromide-stabilized Au nanoparticle core structures onto an indium tin oxide-coated glass slide or silicon wafer, followed by immersion of the functionalized solid support in a solution of 4-aminothiophenol in water/acetonitrile to adsorb the active Raman reporter molecule onto the surface of the Au nanoparticles and subsequent immersion for 1 hour into a suspension of Au nanoparticles with an average diameter of 24 nm. The aggregative state of SERS tags can be monitored by controlling the time of immersion of the functionalized solid support in the suspension of peripheral Au nanoparticles and/or reducing the concentration of peripheral Au nanoparticles in the said suspension. Based on the method described by Ruan et al., lies in the use of a surfactant such as cetyltrimethylammonium bromide to stabilize the Au colloid, which significantly reduces the surface available for the adsorption of an active Raman reporter molecule on the central structure of the Au nanoparticle and thereby the intensity of the SERS signal supplied by SERS. tags. To bind the peripheral Au nanoparticles to the central structure of the Au nanoparticles, the aggregation method involves active Raman reporter molecules having two functional groups with affinity for the Au surface. Therefore, this method is only applicable to the preparation of SERS tags containing active Raman reporter molecules that have two functional groups with affinity for the Au surface, which represents a serious limitation in terms of the application of active Raman reporter molecules as " fingerprint" SERS tag. Moreover, this method requires a long reaction time and is not suitable for cost-effective production of large quantities of SERS tags.

Another method for assembling SERS tags with a narrow size distribution using a solid support was described by Yoon et al. (ACS Nano 2012, 8, 7199 - 7208). The method is based on the size-dependent propensities of Au nanoparticles adsorbed on amine-functionalized glass slides and the use of alkanedithiols to bind the peripheral Au nanoparticles to the central structure of the Au nanoparticle. SERS tags were obtained with 13±3 peripheral Au nanoparticles with an average diameter of 13 nm per central Au nanoparticle structure with an average diameter of 51 nm. The method developed by Yoon and al. appears to make it possible to incorporate any active Raman reporter molecule into a SERS tag. However, this method has limitations in terms of the size of the Au nanoparticles that can be used and the surface area of the central Au nanoparticle structure accessible to active Raman reporter molecules. Moreover, this method requires a long reaction time and is not suitable for cost-effective production of large quantities of SERS tags.

Thus, there remains a need for reproducible, cost-effective and versatile methods for the production of SERS tags that will enable the efficient production of large quantities of SERS tags characterized by a narrow size distribution and a high proportion of low-number aggregates. To obtain increased consistent SERS responses, SERS tags with a large number of low-number aggregates are very necessary.

Brief description of the invention

Accordingly, the object of the present invention is to provide a universal, cost-effective and reproducible method for the production of SERS tags, which makes it possible to expediently obtain large quantities of SERS tags characterized by a narrow size distribution and a high proportion of low-number aggregates. This is achieved by implementing a method for producing surface enhanced Raman spectroscopy (SERS) tags, preferably SERS tags for use as a security element, comprising the following steps:

a) obtaining a first colloid essentially consisting of nanoparticles with a plasmonic surface and essentially the same size, dispersed in an aqueous solvent, and a stabilizing agent adsorbed on the surface of said nanoparticles, and characterized by a ζ -potential value less than or equal to -25 mV;

b) obtaining a second colloid essentially consisting of nanoparticles with a plasmonic surface and essentially the same size, dispersed in an aqueous solvent, active Raman reporter molecules adsorbed on the surface of said nanoparticles, and a stabilizing agent adsorbed on the surface of said nanoparticles, and characterized by a ζ value -potential less than or equal to -25 mV;

c) combining the first colloid with the second colloid such that the ratio of the number of nanoparticles of the first colloid to the number of nanoparticles of the second colloid is from about 25:1 to about 1:1, preferably from about 5:1 to about 1:1, more preferably from 4 : 1 to approximately 3 : 1 to obtain the third colloid;

d) inducing aggregation of nanoparticles using any of stages d1) - d3) or a combination thereof:

d1) mixing the third colloid obtained in step c) at a pH ranging from about 2.2 to the lowest pH at which the active Raman reporter molecules have a net electrical charge of from 0 to 0.3;

d2) adding a salt solution, preferably an inorganic salt solution, to the third colloid obtained in step c);

d3) adding a water-soluble solvent to the third colloid obtained in step c); And

e) stopping aggregation.

Preferably, step b) in the method claimed and described herein comprises the following steps, carried out in the order b1)-b3):

b1) obtaining a colloid essentially consisting of nanoparticles with a plasmonic surface and essentially the same size, dispersed in an aqueous solvent, and a stabilizing agent adsorbed on the surface of these nanoparticles, and characterized by a ζ -potential value less than or equal to -25 mV;

b2) bringing the pH of the colloid to the lowest pH value at which the active Raman reporter molecules that are to be adsorbed on the surface of the nanoparticles carry no net electrical charge, while maintaining the ζ -potential value below or equal to -25 mV, preferably below -40 mV; And

b3) adding a solution of active Raman reporter molecules in a solvent to the colloid obtained in step b2), while maintaining the ζ -potential value below or equal to -25 mV.

In a further preferred embodiment, steps c) and d) are carried out simultaneously in a continuous flow system. In a further preferred embodiment, the size of the nanoparticles in the first colloid is different from the size of the nanoparticles in the second colloid.

As illustrated by Examples E1-E14, the method claimed herein allows for the integration of a number of active Raman reporter molecules into a SERS tag and is not limited to a combination of specific nanoparticle sizes, which also allows the synthesis of SERS tags containing nanoparticles of the same size, which confirmed, for example, by examples E1-E8 and E12-E14. Additionally, the manufacturing method claimed and described herein produces SERS tags with a narrow size distribution, as illustrated, for example, in FIG. 3a and fig. 3b, and a high ratio of low number aggregates, as illustrated, for example, in FIG. 3a and fig. 3b, and is characterized by a significantly shorter reaction time compared to methods known from the literature.

Brief description of drawings

In fig. 1a schematically shows a method for producing SERS tags according to the present invention. The fabrication method involves combining a second colloid (120a) essentially consisting of nanoparticles with a plasmonic surface and substantially the same size (121a) dispersed in an aqueous solvent (not shown), wherein the nanoparticles have active Raman reporter molecules adsorbed on their surface (122a ) and a stabilizing agent (not shown), with the first colloid essentially consisting of nanoparticles with a plasmonic surface and substantially the same size (130a) dispersed in an aqueous solvent (not shown), the nanoparticles having a stabilizing agent (not shown) adsorbed on their surface shown), and induction of nanoparticle aggregation to produce SERS tags (140a).

In fig. 1b is a schematic representation of an embodiment of the method for producing SERS tags of the present invention. In this particular manufacturing method, the continuous flow reactor comprises two pressure vessels (111b, 112b), a tee (113b) and a collection tank (114b), each of the two pressure vessels (111b, 112b) being connected by a tube to tee (113b), which is itself connected by a tube to a collection tank (114b). The manufacturing method involves combining and simultaneously mixing at a pH ranging from about 2.2 to the lowest pH at which the active Raman reporter molecules have a net electrical charge of 0 to 0.3, in a tube connecting the tee (113b) to the reservoir (114b) to collect a continuous flow reactor, a first colloid supplied from a reservoir (112b) under pressure and essentially consisting of nanoparticles with a plasmonic surface and essentially the same size (130b) dispersed in an aqueous solvent (not shown), wherein said the nanoparticles have a stabilizing agent (not shown) adsorbed on their surface, with a second colloid (120b) supplied from a reservoir (111b) under pressure and essentially consisting of nanoparticles with a plasmonic surface and essentially the same size (121b) dispersed in an aqueous solvent ( not shown), wherein said nanoparticles have active Raman reporter molecules (122b) and a stabilizing agent (not shown) adsorbed on their surface. Mixing at a pH ranging from approximately 2.2 to the lowest pH at which active Raman reporter molecules have a net electrical charge of 0 to 0.3 induces nanoparticle aggregation. The aggregation step is stopped in the collection tank (114b), thereby obtaining the target SERS tags (140b).

In fig. 1c is a schematic representation of an embodiment of the method for producing SERS tags of the present invention. In this particular manufacturing method, the continuous flow reactor contains three pressure vessels (111c, 112c, 150c), a tee (113c) and a collection tank (114c). Each of the two pressure reservoirs (111c, 112c) is connected by a tube to a tee (113c), which is itself connected by a tube to a collection reservoir (114c). The manufacturing method involves combining and simultaneously mixing at a pH ranging from approximately 2.2 to the lowest pH at which active Raman reporter molecules have a net electrical charge of 0 to 0.3, in a tube connecting the tee (113c) to the reservoir (114c) to collect a continuous flow reactor, the first colloid supplied from a reservoir (112c) under pressure and essentially consisting of nanoparticles with a plasmonic surface and essentially the same size (130c) dispersed in an aqueous solvent (not shown), wherein the nanoparticles have a stabilizing agent (not shown) adsorbed on their surface, with a second colloid (120c) supplied from a reservoir (111c) under pressure and essentially consisting of nanoparticles with a plasmonic surface and essentially the same size (121c) dispersed in an aqueous solvent (not shown), wherein said nanoparticles have active Raman reporter molecules (122c) and a stabilizing agent (not shown) adsorbed on their surface. Mixing at a pH ranging from approximately 2.2 to the lowest pH at which active Raman reporter molecules have a net electrical charge of 0 to 0.3 induces nanoparticle aggregation. The aggregation step is stopped by introducing the water stored in the pressure tank (150c) into the tube leading into the collection tank (114c) so as to dilute the colloid by introducing the stock solution stored in the pressure tank (150c) into a tube leading to a collection reservoir (114c) by introducing a polymer stored in a reservoir (150c) under pressure into a tube leading into a collection reservoir (114c) or by introducing a precursor dielectric material stored in a reservoir (150c) under pressure into a tube leading to a collection reservoir (114c), thereby obtaining the target SERS tags (140c).

In fig. 2a-c illustrate the change in SERS signal intensity with the aggregation reaction time: FIG. 2a shows the change in SERS intensity with aggregation reaction time for SERS tags prepared as described in example E1; in fig. 2b shows the change in SERS intensity with aggregation reaction time for SERS tags prepared as described in example E14; in fig. 2c shows the variation of SERS intensity with aggregation reaction time for SERS tags prepared as described in example E13. The horizontal axis corresponds to the aggregation reaction time in seconds, and the vertical axis corresponds to the SERS response when excited at 785 nm. Recorded SERS intensity was measured with an LED charge storage time of 100 ms every 1 second at a given gold concentration, and for a given signal intensity was correlated with samples diluted to 12.5 μg Au/mL and scanned with a charge storage time of 1 second. LED.

In fig. 3a-b show SEM images obtained from SERS tags containing nanoparticles of different sizes and manufactured according to example E9 (Fig. 3a) and 10 (Fig. 3b). As confirmed by SEM images, SERS tags produced in accordance with the present invention are enriched in low number aggregates such as dimers, trimers and tetramers and have a narrow size distribution.

Fig. 4 is a graph illustrating the value of the ζ -potential of the first colloid A2 (shown in the graph as filled circles) and the second colloids D2 (shown in the graph as filled triangles), D5 (shown in the graph as filled diamonds) and D6 (shown in the graph as filled squares) . when titrating these colloids with an aqueous solution of 1 N. HCl or 1 N aqueous solution. NaOH. The horizontal axis corresponds to the pH value, and the vertical axis corresponds to the ζ -potential value (mV). Conditions under which aggregation was visible (i.e., the colloids were unstable) are circled. As confirmed by FIG. 4, a number of colloids characterized by a ζ -potential value below -25 mV were stable. Such colloids can be used as the first and second colloids, respectively, in the manufacturing method of the present invention.

In fig. Figure 5 illustrates the development of SERS signal intensity values for colloids aggregating in a flow system. Each measurement was taken at 1-meter marks along the 7-m clear FEP tube. Data, represented by diamonds connected by a solid line, were obtained from SERS tags obtained as described in Example E8. The data represented by circles connected by a dotted line were obtained from SERS tags obtained as described in Example E11.

Detailed description

Definitions

To interpret the meaning of the terms discussed in the description and set forth in the claims, the following definitions should be used.

As used herein, the singular form of object refers to one or more objects and is not necessarily limited to the singular.

As used herein, the term “ at least ” is intended to mean one or more, such as one, or two, or three.

The term “ comprising ” as used herein is non-exclusive and subject to change. Thus, for example, a solution containing compound A may include compounds other than A. However, the term “ comprising ” also covers, as its particular embodiment does, the more restrictive meanings of “ consisting essentially of ” and “ consisting of ” so that, for example, "a solution containing A, B and optionally C" could also (mostly) consist of A and B or (mostly) consist of A, B and C.

When the present disclosure concerns “ preferred ” embodiments/features, combinations of these “ preferred ” embodiments/features are also to be considered disclosed as long as the combination of “ preferred ” embodiments/features is technically meaningful.

As used herein, the term “ about ” means that the specified amount or value may have a specific defined value or some other value adjacent to it. In general, the term " approximately " when denoting a specific value is intended to indicate a range within ±5% of the value. As one example, the phrase "about 100" denotes a range of 100 ± 5, i.e., a range from 95 to 105. Preferably, the range denoted by " about " means a range within ± 3% of the value, more preferably ± 1 %. In general, when the term “ about ” is used, it can be expected that similar results or effects according to the present invention can be obtained within a range of ±5% of the stated value.

Surprisingly, it has been discovered that a method for producing surface enhanced Raman spectroscopy (SERS) tags, preferably SERS tags for use as a security element, involves the following steps:

a) obtaining a first colloid essentially consisting of nanoparticles with a plasmonic surface and essentially the same size, dispersed in an aqueous solvent, and a stabilizing agent adsorbed on the surface of said nanoparticles, and characterized by a ζ -potential value less than or equal to -25 mV;

b) obtaining a second colloid essentially consisting of nanoparticles with a plasmonic surface and essentially the same size, dispersed in an aqueous solvent, active Raman reporter molecules adsorbed on the surface of said nanoparticles, and a stabilizing agent adsorbed on the surface of said nanoparticles, and characterized by a ζ value -potential less than or equal to -25 mV;

c) combining the first colloid with the second colloid such that the ratio of the number of nanoparticles of the first colloid to the number of nanoparticles of the second colloid is from about 25:1 to about 1:1, preferably from about 5:1 to about 1:1, more preferably from about 4:1 to approximately 3:1 to obtain the third colloid;

d) inducing aggregation of nanoparticles using any of stages d1) - d3) or a combination thereof:

d1) mixing the third colloid obtained in step c) at a pH ranging from about 2.2 to the lowest pH at which the active Raman reporter molecules have a net electrical charge of from 0 to 0.3;

d2) adding a salt solution, preferably an inorganic salt solution, to the third colloid obtained in step c);

d3) adding a water-soluble solvent to the third colloid obtained in step c); And

e) stopping aggregation;

produces in a cost-effective and expedient manner a large number of SERS tags with a narrow size distribution and a high ratio of low-number aggregates. The method does not have any limitations in terms of the active Raman reporter molecule to be used as the fingerprint of the SERS tag or the size of the plasmonic nanoparticles contained in the first and second colloid, allowing the fabrication of various SERS tags.

As is well known to one skilled in the art and used herein, a SERS tag comprises a single nanoparticle aggregate with a plasmonic surface and active Raman reporter molecules adsorbed on the surface of the nanoparticles. The plasmonic surface nanoparticles are responsible for generating the electric field required for Raman enhancement, while the active Raman reporter molecules provide the unique vibrational fingerprint of the SERS tag. The SERS tag may additionally contain an outer coating layer that isolates the aggregate of nanoparticles with active Raman scattering molecules adsorbed on the surface from the external environment. Thus, the outer coating layer a) isolates the SERS tag from the external environment, thereby preventing the leaching of active Raman reporter molecules from the SERS tag and protecting the SERS tag from environmental contamination that can cause the appearance of false peaks, b) increases colloidal stability of the SERS tag and c) provides a convenient surface for further chemical functionalization. The outer coating layers include silica and polymers such as polyethylenimine (PEI), sodium styrene maleic acid copolymer (PSMA), polydiallyldimethylammonium chloride (PDADMAC).

Due to the emerging SERS signal, SERS tags obtained using the manufacturing method claimed and described herein are particularly suitable as a security element for protecting documents and products from counterfeiting and illegal reproduction. As used herein, the term " security element " means an element that may be included in or applied to a security document or article for the purpose of determining its authenticity and protecting it from counterfeiting and illegal reproduction. The security element may be a mark, image, design or graphic element printed, coated or sprayed on the security element or the article with an ink, varnish or coating composition that contains SERS tags obtained using the manufacturing method of the present invention. Alternatively, SERS tags can function as a security element if they are embedded in the underlying document. The terms " security document " and " security article " refer to a document or article that has a value that makes it potentially susceptible to attempts at counterfeiting or unauthorized reproduction, and that is generally protected against forgery or falsification by at least one security feature. The term " secured article " in the context of this document covers all products that must be protected against counterfeiting and/or illegal reproduction in order to guarantee their contents. Examples of protected documents include, but are not limited to, valuable documents and valuable commercial items. Typical examples of valuable documents include, but are not limited to, banknotes, legal documents, tickets, checks, vouchers, revenue stamps, tax stamps, agreements, etc., identification documents such as passports, identity cards, visas, bank cards, credit cards , transaction cards, access documents, entrance tickets, etc. High-value commercial goods cover packaging material, in particular for cosmetic products, nutraceutical products, pharmaceutical products, alcoholic beverages, tobacco products, beverages or food products, electrical/electronic products, textiles or jewelry, i.e. products that must be protected against counterfeiting and/or illegal reproduction to guarantee the validity of the contents of the packaging, similar to, for example, genuine medicinal products. Examples of packaging materials include, but are not limited to, labels such as product authentication labels, tamper evident labels and seals.

In the context of the present invention, by definition, the term “ nanoparticle ” means a single particle having a size corresponding to a maximum physical size (e.g., length, diameter, etc.) in the range of 20 ± 5 nm to 160 ± 5 nm, preferably from 40 ± 5 nm to 140 ± 5 nm. The nanoparticle in the context of the present invention has a plasmonic surface, that is, the nanoparticle has an outer surface capable of enhancing the Raman scattering of the active Raman molecule. The outer surface of the nanoparticles is made of 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 gold (Au ). The nanoparticle in the context of the present invention can be solid or hollow and is preferably solid. A solid nanoparticle can be made of a single material, i.e., the SERS-enhancing material on the outer surface of the nanoparticle, or from several materials, i.e., the material(s) of the central structure of the nanoparticle may be different from the SERS-enhancing material on the outer surface surface of the nanoparticle. A hollow nanoparticle is a nanoparticle whose central structure is an empty space. The nanoparticle can have any shape that can be obtained. Preferably, the nanoparticle is a solid Au nanoparticle. Preferably, the nanoparticle has a shape selected from the group consisting of sphere, spheroid, rod, disk, prism and cube, more preferably selected from sphere and spheroid, and even more preferably the nanoparticle has a spheroid shape.

In the context of this document, the wording “nanoparticles with essentially the same size" means that said nanoparticles have a size corresponding to a maximum physical size (e.g. length, diameter, etc.) within ± 20 nm, preferably within ± 10 nm, of the average size determined for said nanoparticles using electron microscopy techniques such as transmission electron microscopy (TEM) or scanning electron microscopy (SEM), or, if applicable, using a published method Haiss et al. (Anal. Chem. 2007,79, 4215-4221).

Nanoparticles of the first colloid, the second colloid and the colloid obtained 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-soluble solvents, wherein the water-soluble solvent is preferably selected from the list including methanol, ethanol, propanol, isopropanol, tetrahydrofuran, N -methyl-2-pyrrolidone (NMP ), dimethyl sulfoxide (DMSO), N , N -dimethylformamide (DMF), acetone and acetonitrile.

The first colloid obtained in manufacturing method step a) and the second colloid obtained in manufacturing method step b) as well as the colloid obtained in manufacturing method step b1) do not contain polymers or surfactants for stabilization. Stabilization of colloids with polymers (eg polyvinylpyrrolidone) or surfactants (eg cetyltrimethylammonium bromide) is well known in the art. However, because polymers and surfactants are added to colloids prior to the addition of active Raman reporter molecules, they significantly reduce the surface area available on the nanoparticles for adsorption of active Raman reporter molecules and increase the distance between colloidal particles in the aggregate, which is how As a result, SERS tags exhibit a lower SERS signal intensity. To overcome this disadvantage, the first colloid, the second colloid, and the colloid obtained in step b1) do not contain surfactants and polymers.

The stabilizing agent adsorbed on the surface of the nanoparticles of the first colloid, the second colloid and the colloid obtained in step b1) 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 active Raman reporter molecules and the stabilizing agent during adsorption of the active Raman reporter molecules onto the surface of the nanoparticles, it is preferable that the stabilizing agent does not contain groups having an affinity for SERS-enhancing materials, particularly gold. Examples of such groups are 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, salts of carboxylic acids, ascorbic acid, salts of ascorbic acid 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, dipotassium hydrogen citrate, and tripotassium citrate. Lactic acid salts include sodium lactate and potassium lactate. Salts of ascorbic acid include sodium ascorbate and potassium ascorbate. In the most 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, as well as mixtures thereof.

Advantageously, the manufacturing method claimed and described herein allows for the integration of any active Raman reporter molecule into a SERS tag. Preferred active Raman reporter molecules include fully conjugated molecules containing an aryl group substituted with one or more substituents selected from NR ¹ R ² , -SH, -≡, -≡N and -N═, preferably from -NR ¹ R ² and -SH, and/or an N -containing heteroaryl group, and/or an S -containing heteroaryl group, wherein the radicals R ¹ and R ² are independently selected from -H and alkyl, preferably from -H and C ₁ -C ₄ -alkyl.

As is known to one skilled in the art of organic chemistry, a fully conjugated molecule is a molecule having a system of conjugated electrons spanning the entire molecule. A conjugated electron system is a system of coupled p-orbitals with delocalized electrons.

As is well known to one skilled in the art, an " aryl group " is a group derived from a monocyclic or polycyclic aromatic hydrocarbon compound by the removal of one hydrogen atom from the ring carbon atom. 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 cyclic conjugated π system. As part of a cyclic conjugated π system, the S -containing heteroaromatic compound may additionally 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 cyclic conjugated π system. As part of a cyclic conjugated π system, the N -containing heteroaromatic compound may additionally contain one or more oxygen atoms. Examples of N -containing heteroaryl groups include, but are not limited to, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, benzoimidazolyl, indazolyl, benzotriazolyl, pyridinyl, pyrimidinyl, pyridazinyl, triazinyl, quinolinyl, isoquinolinyl, diazanfatyl, quinalosinyl, cinnolinyl, ft alasinyl, quinoxalinyl, purinyl, azaphenanthryl, diazaphenanthryl , azaanthracenyl, diazaanthracenyl, azapyrenyl, diazapyrenyl, oxazolyl, isoxazolyl, oxadiazolyl, benzoxazolyl, benzisoxazolyl and benzoxadiazolyl.

Preferred active Raman reporter molecules include, but are not limited to, the following:

- a fully conjugated compound consisting of an aryl group substituted by one or more substituents selected from -NR ¹ R ² , -SH, -≡, -≡N and -N═, preferably from -NR ¹ R ² and -SH, which connected directly or via a linker -L ¹ - to an aryl group substituted by one or more substituents selected from the list including an amino group (-NH ₂ ), an N -alkylamino group, an N , N -dialkylamino group, a thiol group, an ethynyl group, a cyano group and an isocyanide group a group, an N -containing heteroaryl group or an S -containing heteroaryl group, wherein

the substituents R ¹ and R ² have the meanings as defined herein;

linker -L ¹ - selected from -CR ⁸ ═CR ⁹ -, -N=N-, -≡-, -CR ¹⁰ ═CR ¹¹ - o -C ₆ H ₄ -, -CR ¹⁰ ═CR ¹¹ - m -C ₆ H ₄ -, -CR ¹⁰ ═CR ¹¹ - p -C ₆ H ₄ -, -CR ¹⁰ ═CR ¹¹ - o -C ₆ H ₄ -CR ¹² ═CR ¹³ -, -CR ¹⁰ ═CR ¹¹ - m - C ₆ H ₄ -CR ¹² ═CR ¹³ -, -CR ¹⁰ ═CR ¹¹ - p -C ₆ H ₄ -CR ¹² ═CR ¹³ -, -CR ¹⁴ ═NN═CR ¹⁵ -,

, , , , , , , , , , , , , , , , , , , , , , And ;

And

the substituents R ⁸ - R ¹⁵ 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, which is connected directly or via a linker -L ¹ - to an N -containing heteroaryl group or an S -containing heteroaryl group,

wherein

linker -L ¹ - selected from -CR ⁸ ═CR ⁹ -, -N=N-, -≡-, -CR ¹⁰ ═CR ¹¹ - o -C ₆ H ₄ -, -CR ¹⁰ ═CR ¹¹ - m -C ₆ H ₄ -, -CR ¹⁰ ═CR ¹¹ - p -C ₆ H ₄ -, -CR ¹⁰ ═CR ¹¹ - o -C ₆ H ₄ -CR ¹² ═CR ¹³ -, -CR ¹⁰ ═CR ¹¹ - m - C ₆ H ₄ -CR ¹² ═CR ¹³ -, -CR ¹⁰ ═CR ¹¹ - p -C ₆ H ₄ -CR ¹² ═CR ¹³ -, -CR ¹⁴ ═NN═CR ¹⁵ -,

, , , , , , , , , , , , , , , , , , , , , , And ;

And

the substituents R ⁸ - R ¹⁵ 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, which is connected directly or via a linker -L ¹ - to an S -containing heteroaryl group,

wherein

linker -L ¹ - selected from -CR ⁸ ═CR ⁹ -, -N=N-, -≡-, -CR ¹⁰ ═CR ¹¹ - o -C ₆ H ₄ -, -CR ¹⁰ ═CR ¹¹ - m -C ₆ H ₄ -, -CR ¹⁰ ═CR ¹¹ - p -C ₆ H ₄ -, -CR ¹⁰ ═CR ¹¹ - o -C ₆ H ₄ -CR ¹² ═CR ¹³ -, -CR ¹⁰ ═CR ¹¹ - m - C ₆ H ₄ -CR ¹² ═CR ¹³ -, -CR ¹⁰ ═CR ¹¹ - p -C ₆ H ₄ -CR ¹² ═CR ¹³ -, -CR ¹⁴ ═NN═CR ¹⁵ -,

, , , , , , , , , , , , , , , , , , , , , , And ;

and the substituents R ⁸ - R ¹⁵ 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 -NR ¹ R ² , -SH, -≡, -≡N and -N═, preferably from -NR ¹ R ² and -SH, N -containing heteroaryl group, optionally substituted with one or more substituents selected from -NR ³ R ⁴ , -SH, -≡, -≡N and -N═, or S -containing heteroaryl group, optionally substituted one or more substituents selected from -NR ⁵ R ⁶ , -SH, -≡, -≡N and -N═ attached directly to a hydrogen atom, wherein the substituents R ¹ and R ² have the meanings defined herein, and the substituents R ³ - R ⁶ are independently selected from -H and alkyl, preferably from -H and C ₁ -C ₄ -alkyl.

An aryl group substituted with one or more substituents selected from -NR ¹ R ² , -SH, -≡, -≡N and -N═, preferably from -NR ¹ R ² and -SH, may contain one or more additional substituents, preferably selected from hydroxy, alkyl, alkoxy, alkylthio, formyl, nitro, halide, hydroxycarbonyl, alkoxycarbonyl and O -containing heteroaryl group and more preferably selected from alkyl, alkoxy, alkylthio, halides and O -containing heteroaryl group.

The N -containing heteroaryl group and the S -containing heteroaryl group may contain one or more additional substituents, preferably selected from amino, N -alkylamino, N , N -dialkylamino, thiol, hydroxy, alkyl, alkoxy, alkylthio, formyl, cyano, isocyanide, ethynyl, nitro, halide, hydroxycarbonyl, alkoxycarbonyl and O -containing heteroaryl group, preferably from alkyl, alkoxy, alkylthio, halides and O -containing heteroaryl group.

Examples of O -containing heteroaryl groups include, but are not limited to, furanyl, benzofuranyl, isobenzofuranyl, oxazolyl, isoxazolyl, benzoxazolyl, and oxadiazolyl.

A preferred active Raman reporter compound is a compound of general formula (I),

wherein

A ¹ , B ¹ and C ¹ are independently selected from N, CR ¹⁶ and CR ¹⁷ , with the proviso that only one of A ¹ , B ¹ and C ¹ is N;

A ² , B ² and C ² are independently selected from N, CR ¹⁸ and CR ¹⁹ , with the proviso that only one of A ² , B ² and C ² is N;

E ¹ , D ¹ , E ² , D ² , R ¹⁶ , R ¹⁷ , R ¹⁸ and R ¹⁹ are independently selected from hydrogen, amino, N -alkylamino, N , N -dialkylamino, thiol, hydroxy, alkyl, alkoxy , alkylthio, formyl, cyano, isocyanide, alkynyl, nitro, halide, hydroxycarbonyl, alkoxycarbonyl and O -containing heteroaryl group, preferably from hydrogen, alkyl, alkoxy, alkylthio, halides and O -containing heteroaryl group; And

X represents a single bond or linker -L ² - selected from -CR ⁸ ═CR ⁹ -, -N=N-, -≡-, -CR ¹⁰ ═CR ¹¹ - o -C ₆ H ₄ -, -CR ¹⁰ ═CR ¹¹ - m -C ₆ H ₄ -, -CR ¹⁰ ═CR ¹¹ - p -C ₆ H ₄ -, -CR ¹⁰ ═CR ¹¹ - o -C ₆ H ₄ -CR ¹² ═CR ¹³ -, -CR ¹⁰ ═CR ¹¹ - m -C ₆ H ₄ -CR ¹² ═CR ¹³ -, -CR ¹⁰ ═CR ¹¹ - p -C ₆ H ₄ -CR ¹² ═CR ¹³ -, -CR ¹⁴ ═NN═CR ¹⁵ -,

, , , , , , , , , , , , , , , , , , , , , , And ;

wherein R ⁸ - R ¹⁵ is selected from hydrogen, alkyl, alkoxy, alkylthio, formyl, cyano, nitro, halide, hydroxycarbonyl and alkoxycarbonyl.

Preferably, the residues A ¹ and A ² represent N in the general formula (I). More preferably, the residues A ¹ and A ² represent N and the substituents E ¹ , D ¹ , E ² , D ² , R ¹⁶ , R ¹⁷ , R ¹⁸ and R ¹⁹ represent hydrogen in the general formula (I).

Active Raman reporter molecules include, but are not limited to, the following: 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-tetrazole-5-thiol, 2-fluorothiophenol, 2-naphthalenethiol, 4-(((3-mercapto-5-( 2-methoxyphenyl)-4H-1,2,4-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)-4H-1,2,4-triazol-4-yl) imino)methyl)-1,2-benzenediol, 4-(((3-mercapto-5-(2-pyridinyl)-4H-1,2,4-triazol-4-yl)imino)methyl)benzoic acid, 2 ,3,4,6-tetrafluorobenzenethiol, (5-(4-methoxyphenyl)-1,3,4-oxidazol-2-thiol), ( E )-1,2-di(pyridin-4-yl)ethene, 5 -(pyridin-4-yl)-1,3,4-oxadiazol-2-thiol and 1,4-bis(( E )-2-(pyridin-4-yl)vinyl)benzene.

The first colloid obtained in step a) is characterized by a ζ-potential value less than or equal to -25 mV. The second colloid obtained 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 of nanoparticle material/mL using a Malvern Zetasizer Nano-ZS with 1-mL curved capillary cuvettes. If necessary, namely in the case of colloids with a concentration higher than 0.05 mg of nanoparticle material/ml, before measuring the ζ -potential, the colloid is diluted with deionized water to achieve a concentration of nanoparticle material of 0.05 mg/ml.

Preferably, the concentration of nanoparticle material (mg/ml) in the first colloid and in the second colloid is below 0.66 mg/ml and more preferably is from about 0.05 mg/ml to 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 such that the ratio of the number of nanoparticles of the first colloid to the number of nanoparticles of the second colloid is from about 25:1 to about 1:1, preferably from about 5:1 to about 1:1, more preferably from about 4:1 to about 3:1, nanoparticle aggregation is induced. The aggregation described herein consists of selective aggregation of nanoparticles of the second colloid with nanoparticles of the first colloid, i.e., there is no aggregation of nanoparticles of the first colloid with nanoparticles of the first colloid, and there is no or little aggregation of nanoparticles of the second colloid with nanoparticles of the second colloid.

Aggregation of nanoparticles is induced using any of stages d1) - d3) or a combination thereof:

d1) mixing the third colloid obtained in step c) at a pH ranging from about 2.2 to the lowest pH at which the active Raman reporter molecules have a net electrical charge of from 0 to 0.3;

d2) adding a salt solution, preferably an inorganic salt solution, to the third colloid obtained in step c);

d3) adding a water-soluble solvent to the third colloid obtained in step c).

In a preferred embodiment, induction of nanoparticle aggregation involves step d1), namely mixing the third colloid obtained in step c), at a pH ranging from about 2.2 to the lowest pH at which the active Raman reporter molecules have a net electrical charge from 0 to 0.3. The net electrical charge of the active Raman reporter molecule and the pH value corresponding to this net electrical charge can be predicted using online tools such as Chemicalize.com (Chemicalize. ChemAxon. http://chemicalize.com/#/calculation). At a pH in the range of about 2.2 to the lowest pH at which the active Raman reporter molecules have a net electrical charge of 0 to 0.3, selective aggregation of the first colloid nanoparticles with the second colloid nanoparticles occurs.

Alternatively, inducing nanoparticle aggregation involves step d2) adding a salt solution, preferably an inorganic salt solution, to the third colloid obtained in step c). Salt-induced aggregation of nanoparticles is a well-known method for colloid chemistry specialists to induce nanoparticle aggregation ( ChemPhysChem 2018, 19 , 24 - 28). Examples of inorganic salts that need to be used in step d2) of the manufacturing method, in the form of a solution such as an aqueous solution, include, but are not limited to, sodium fluoride, sodium chloride, sodium bromide, sodium iodide, magnesium chloride, potassium chloride and mixtures thereof.

In a further alternative embodiment, the induction of nanoparticle aggregation involves step d3), namely the addition of a water-soluble solvent to the third colloid obtained in step c). The water-soluble solvent is preferably selected from the list including methanol, ethanol, propanol, isopropanol, tetrahydrofuran, N -methyl-2-pyrrolidone (NMP), dimethyl sulfoxide (DMSO), N , N -dimethylformamide (DMF), acetone and acetonitrile.

The preferred manufacturing method of the present invention involves the following steps:

a) obtaining a first colloid essentially consisting of nanoparticles with a plasmonic surface and essentially the same size, dispersed in an aqueous solvent, and a stabilizing agent adsorbed on the surface of said nanoparticles, and characterized by a ζ -potential value less than or equal to -25 mV;

b) obtaining a second colloid essentially consisting of nanoparticles with a plasmonic surface and substantially the same size dispersed in an aqueous solvent, an active Raman reporter molecule of general formula (I) adsorbed on the surface of said nanoparticles, and a stabilizing agent adsorbed on the surface of said nanoparticles , and characterized by a ζ -potential value below or equal to -25 mV;

c) combining the first colloid with the second colloid such that the ratio of the number of nanoparticles of the first colloid to the number of nanoparticles of the second colloid is from about 25:1 to about 1:1, preferably from about 5:1 to about 1:1, more preferably from about 4:1 to approximately 3:1 to obtain the third colloid;

d) inducing aggregation of nanoparticles using any of stages d1) - d3) or a combination thereof:

d1) mixing the third colloid obtained in step c) at a pH ranging from about 2.2 to about 6.1, preferably from about 2.6 to about 5.7;

d2) adding a salt solution, preferably an inorganic salt solution, to the third colloid obtained in step c);

d3) adding a water-soluble solvent to the third colloid obtained in step c); And

e) stopping aggregation.

Preferably, in the manufacturing methods described herein, step b) comprises the following steps, carried out in the order b1) to b3):

b1) obtaining a colloid containing nanoparticles with a plasmonic surface and essentially the same size, dispersed in an aqueous solvent, and a stabilizing agent adsorbed on the surface of these nanoparticles, and characterized by a ζ -potential value less than or equal to -25 mV;

b2) bringing the pH of the colloid to a value greater than the lowest pH value at which the active Raman reporter molecules that are to be adsorbed on the surface of the nanoparticles carry no net electrical charge, while maintaining the ζ -potential value below or equal to -25 mV, preferably below -40 mV; And

b3) adding a solution of Raman active molecules in a solvent to the colloid obtained in step b2), while maintaining the ζ -potential value below or equal to -25 mV.

The colloid obtained in step b1) of the manufacturing method claimed herein actually consists of nanoparticles with a plasmonic surface and substantially the same size dispersed in an aqueous solvent, and a stabilizing agent adsorbed on the surface of said nanoparticles. The first colloid obtained in step a) of the manufacturing method claimed herein actually consists of nanoparticles with a plasmonic surface and substantially the same size dispersed in an aqueous solvent, and a stabilizing agent adsorbed on the surface of said nanoparticles. The second colloid obtained in step b) of the manufacturing method claimed herein essentially consists of nanoparticles with a plasmonic surface and substantially the same size dispersed in an aqueous solvent, active Raman reporter molecules adsorbed on the surface of said nanoparticles, and a stabilizing agent adsorbed on the surface of these nanoparticles. Thus, the nanoparticles of the first colloid and the nanoparticles of the second colloid do not carry on their surface molecules or organisms that provide a specific interaction through a “key-to-lock” matching mechanism between the nanoparticles of the first colloid and the nanoparticles of the second colloid. Examples of such molecules include antibodies, proteins, antigens, complementary strands of DNA and complementary strands of RNA. Examples of such organisms include bacteria, viruses and spores. Thus, the first colloid, the second colloid, and the colloid obtained in step b1), in addition to being free of surfactants and polymers, are also free of molecules such as antibodies, proteins, antigens, complementary DNA strands and complementary strands of RNA, as well as organisms such as bacteria, viruses and spores.

In step b2) of the manufacturing method of the present invention, the pH of the colloid is adjusted to a value greater than the lowest pH value at which the active Raman reporter molecules to be adsorbed on the surface of the nanoparticles carry no net electrical charge, while maintaining the ζ -potential value below or equal to -25 mV, preferably below -40 mV. The net electrical charge of the active Raman reporter molecule and the pH value corresponding to this net electrical charge can be predicted using online tools such as Chemicalize.com (Chemicalize. ChemAxon. http://chemicalize.com/#/calculation).

Preferably, in step b2) the pH of the colloid is adjusted to a range of about 8.2 to about 12.1 while maintaining the ζ potential value below or equal to -25 mV, preferably below -40 mV. In a more preferred embodiment, the pH of the colloid is adjusted in step b2) to a value of approximately 11.0.

In step b3) of the present invention, a solution of the active Raman reporter molecule in a solvent is added to the colloid obtained in step b2), while maintaining the ζ potential value below or equal to -25 mV. The solvent used to prepare the active Raman reporter molecule solution includes any aqueous solvent and any organic solvent suitable for dissolving the active Raman reporter molecule. Examples of organic solvents include, but are not limited to, alcohols, 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-soluble solvents such as methanol, ethanol, propanol, isopropanol, tetrahydrofuran, N -methyl-2-pyrrolidone (NMP), dimethyl sulfoxide (DMSO),
N , N -dimethylformamide (DMF), acetone and acetonitrile.

The preferred manufacturing method of the present invention involves the following steps:

a) obtaining a first colloid essentially consisting of nanoparticles with a plasmonic surface and essentially the same size, dispersed in an aqueous solvent, and a stabilizing agent adsorbed on the surface of said nanoparticles, and characterized by a ζ -potential value less than or equal to -25 mV;

b) obtaining a second colloid essentially consisting of nanoparticles with a plasmonic surface and substantially the same size dispersed in an aqueous solvent, an active Raman reporter molecule of general formula (I) adsorbed on the surface of said nanoparticles, and a stabilizing agent adsorbed on the surface of said nanoparticles , and characterized by a ζ -potential value below or equal to -25 mV;

c) combining the first colloid with the second colloid such that the ratio of the number of nanoparticles of the first colloid to the number of nanoparticles of the second colloid is from about 25:1 to about 1:1, preferably from about 5:1 to about 1:1, more preferably from 4 : 1 to approximately 3 : 1 to obtain the third colloid;

d) inducing aggregation of nanoparticles using any of stages d1) - d3) or a combination thereof:

d1) mixing the third colloid obtained in step c) at a pH ranging from about 2.2 to about 6.1, preferably from about 2.6 to about 5.7;

d2) adding a salt solution, preferably an inorganic salt solution, to the third colloid obtained in step c);

d3) adding a water-soluble solvent to the third colloid obtained in step c); And

e) stopping aggregation,

in this case, stage b) provides for the following stages, carried out in the order b1)-b3):

b1) obtaining a colloid essentially consisting of nanoparticles with a plasmonic surface and essentially the same size, dispersed in an aqueous solvent, and a stabilizing agent adsorbed on the surface of these nanoparticles, and characterized by a ζ -potential value less than or equal to -25 mV;

b2) adjusting the pH of the colloid to a value in the range of from about 8.0 to about 12.1, preferably from about 8.2 to 12.1, while maintaining the ζ potential value below or equal to -25 mV, preferably below -40 mV ; And

b3) adding a solution of the active Raman reporter molecule of general formula (I) in a solvent to the colloid obtained in step b2), while maintaining the ζ -potential value below or equal to -25 mV.

The present inventors have discovered that the signal intensity of SERS tags made in accordance with the present invention can be further increased by allowing a submonolayer or monolayer of active Raman reporter molecules to be adsorbed onto the surface of nanoparticles contained in a second colloid. Thus, a further preferred embodiment of the present invention relates to a method for making SERS tags as claimed herein, wherein the second colloid nanoparticles have a submonolayer or monolayer of active Raman reporter molecules absorbed on their surface. As used herein, a monolayer of active Raman reporter molecules adsorbed on the surface of nanoparticles of a second colloid refers to a layer of one active Raman reporter molecule thickness adsorbed on the surface of said nanoparticles. As used herein, a submonolayer of active Raman reporter molecules refers to an incomplete monolayer of active Raman reporter molecules. To ensure that a submonolayer or monolayer of active Raman reporter molecules is adsorbed onto the surface of the nanoparticles of the second colloid, the amount of active Raman reporter molecules added in step b3) to the colloid obtained in step b2) must be calculated using methods well known to one skilled in the art. the field of technology of methods that take into account the dependence on the shape and size of nanoparticles of the second colloid.

The manufacturing method disclosed herein also allows for the production of SERS tags containing a mixture of different active Raman reporter molecules, ie, a mixture of two or more different active Raman reporter molecules. This is particularly advantageous in that it provides access to a range of SERS tags, each of which is characterized by a unique SERS signal, by combining in varying ratios a limited number of different active Raman reporter molecules. To obtain such SERS tags, the second colloid nanoparticles obtained in step b) are prepared so that a mixture of different active Raman reporter molecules (i.e., a mixture of two or more different active Raman reporter molecules) is absorbed on their surface. . Such a second colloid can be prepared by step b3) of the method of preparing a solution of active Raman molecules in a solvent containing two or more different active Raman reporter molecules, or by performing step b3) of the method sequentially and each time using a different solution containing a different active Raman reporter molecule. Thus, a further embodiment of the present invention relates to a manufacturing method in which, in step b3), a solution of active Raman reporter molecules in a solvent contains a mixture of two or more different active Raman reporter molecules. Another further embodiment of the present invention relates to a manufacturing method in which step b3) is carried out sequentially n times, with n≥2, each time using a solution containing an active Raman reporter molecule that is structurally different from the active Raman reporter molecules , used in the remaining n-1 solutions, used in the remaining n-1 stages. As used herein, different active Raman reporter molecules refer to active Raman reporter molecules having different chemical structures and providing a different SERS spectrum.

To further increase the number of low-number aggregates and, consequently, the intensity of the SERS signal provided by SERS tags manufactured using the manufacturing method claimed herein, it is preferable that in step c) the ratio of the number of nanoparticles of the first colloid to the number of nanoparticles of the second colloid ranged from about 5:1 to about 1:1, preferably from about 4:1 to about 3:1. As demonstrated, for example, by Example 10 and FIG. 3b, a ratio of first colloid nanoparticles to second colloid nanoparticles of about 5:1 to about 1:1 provides access to SERS tags with a high number of low number aggregates such as dimers, trimers, and tetramers.

To induce aggregation using the method described for step d1), it is necessary to mix the third colloid obtained in step c) at a pH range from about 2.2 to the lowest pH value at which the active Raman reporter molecules have a net electrical charge of 0 to 0.3, which in the case of the active Raman reporter molecule of general formula (I) is a pH in the range of about 2.2 to about 6.1, preferably from about 2.6 to about 5.7. This can be achieved either by adding an acidic solution to the third colloid obtained in step c) or by adjusting the pH of the first colloid to such a value that the colloid obtained in step d1) has the required pH value.

Thus, one embodiment of the present invention relates to a manufacturing method in which step d1) further comprises adding an acidic solution to the third colloid obtained in step c), with stirring, so that the pH of the resulting colloid is in the range of about 2.2 to the lowest pH at which the active Raman reporter molecules have a net electrical charge of from 0 to 0.3, which in the case of the active Raman reporter molecule of general formula (I) is a pH in the range of about 2.2 to about 6 ,1, preferably from about 2.6 to about 5.7. Suitable acidic solutions include, but are not limited to, acetic acid, hydrochloric acid and nitric acid.

To avoid an additional manufacturing step, it is convenient to adjust the pH of the first colloid to such a value that the colloid produced in step d1) has a pH in the range of about 2.2 to the lowest pH at which the active Raman reporter molecules have a net electrical charge from 0 to 0.3, which in the case of the active Raman reporter molecule of general formula (I) is a pH in the range from about 2.2 to about 6.1, preferably from about 2.6 to about 5.7.

The manufacturing method claimed herein also includes the step e) of stopping aggregation. Preferably, step e) comprises any of the following steps e1) - e4):

e1) adjusting the pH of the colloid obtained in step d) to a value greater than the lowest pH value at which the active Raman reporter molecules that are to be adsorbed on the surface of the nanoparticles carry no net electrical charge;

e2) diluting the colloid obtained in step d) with water, preferably so that the concentration of nanoparticles in the colloid becomes below 6*10 ⁹ nanoparticles/ml;

e3) adding the polymer to the colloid obtained in step d);

e4) adding a dielectric material precursor to the colloid obtained in step d).

In the manufacturing method of the present invention as claimed herein, aggregation can be stopped by any of the methods e1) to e4).

Raising the pH of the colloid obtained in step d) to a value greater than the lowest pH value at which the active Raman reporter molecules that are to be adsorbed on the surface of the nanoparticles do not carry a net electrical charge, as described in step e1), resulting in an increase in the number of electrostatic repulsions between nanoparticle aggregates, leading to a stop in the aggregation process. The net electrical charge of the active Raman reporter molecule and the pH value corresponding to this net electrical charge can be predicted using online tools such as Chemicalize.com (Chemicalize. ChemAxon. http://chemicalize.com/#/calculation).

Addition of the polymer to the colloid obtained in step d) as described for step e3) increases the number of steric repulsions between the nanoparticle aggregates and causes the aggregation process to stop. It is advantageous that the polymer used in step e3) does not affect the SERS signal from the SERS tag. Suitable polymers include, but are not limited to, polyvinylpyrrolidone and polyethylene glycol.

Addition of a dielectric material precursor to the colloid obtained in step d) as described in step e4) results in encapsulation of nanoparticle aggregates with at least one layer of dielectric material, which stabilizes the SERS tags and ultimately stops the aggregation process. Preferably, the dielectric material precursor is a silica precursor. Silica precursors include, but are not limited to, solutions of tetraethylorthosilicate and 3-aminopropyltrimethoxysilane in ethanol, solutions of tetraethylorthosilicate 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, aggregation is stopped by diluting the colloid obtained in step d) with water so that a concentration of nanoparticle material [μg/ml] is achieved that is less than or equal to 12.5 μg/ml (see, for example: E1 - E8, E11 - E14). Alternatively, aggregation is stopped by diluting the colloid obtained in step d) with water so that the concentration of nanoparticles falls below 6*10 ⁹ nanoparticles/ml.

A further preferred embodiment of the present invention relates to a method of manufacturing SERS tags, wherein step e) essentially consists of step e3), and the manufacturing method further comprises diluting the colloid obtained in step e3) with water and/or coating the SERS tags dielectric material.

In a further preferred embodiment, steps c) and d) of the manufacturing method claimed herein are carried out simultaneously in a continuous flow system. This particular manufacturing method uses a continuous flow reactor. Such a continuous flow reactor is shown schematically in FIG. 1b and fig. 1c and consists of two pressure reservoirs (111b, 112b, 111c, 112c) connected by a tube to a tee (113b, 113c) and a collection reservoir (114b, 114c). The reservoir (111b, 111c) stores the second colloid under pressure, and the reservoir (112b, 112c) stores the first colloid under pressure. An atmospheric vessel (114b, 114c) is used to collect SERS tags and optionally stop aggregation (114c). A tube connecting each of the pressure reservoirs (111b, 112b, 111c, 112c) to a tee (113b, 113c) is used to transport colloids. An additional tube connecting the tee (113b, 113c) to the collection reservoir (114b, 114c) is used to perform aggregation. The nanoparticle aggregates resulting from the aggregation are collected in a collection vessel (114b) where the aggregation is stopped.

When carrying out steps c) and d) of the manufacturing process simultaneously in a continuous flow system, it is further preferred that step e) is also carried out in a continuous flow system.

As mentioned above, the nanoparticles used in the method of making SERS tags can have any shape that can be obtained, such as sphere, spheroid, rod, disk, prism and cube. Preferably, the shape of the plasmonic surface nanoparticles used in the manufacturing method of the present invention as claimed herein is selected from sphere and spheroid. Even more preferably, said nanoparticles have a spheroid shape.

In a preferred embodiment, the nanoparticles of the first colloid and the nanoparticles of the second colloid are the same size. Conveniently, in this method of producing SERS tags, the colloid used to prepare the second colloid (ie, the colloid obtained 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 SERS tag structures similar to those of the SERS tags depicted in FIG. 3a, or the size of the nanoparticles in the first colloid may exceed the size of the nanoparticles in the second colloid, resulting in the SERS tags having structures similar to those of the SERS tags depicted in FIG. 3b.

The fabrication method reported herein is preferably carried out with colloids, wherein the plasmonic surface of the nanoparticles in the first colloid and/or the plasmonic surface of the nanoparticles in the second colloid consists of gold, more preferably with colloids containing solid gold nanoparticles, and even more preferably with gold colloids, stabilized with citrate.

Examples

The present invention will now be described in more detail in accordance with the following non-limiting examples.

a common part

The reagents described below were obtained from the following suppliers:

Gold chloraurate trihydrate (>99.9%; CAS No. 16961-25-4), sodium borohydride (99.99%; CAS No. 16940-66-2), sodium citrate tribasic dihydrate (≥99.5%; CAS No. 6132 -04-3), ( E )-1,2-di(pyridin-4-yl)ethene (97%; CAS No. 13362-78-2), 5-(pyridin-4-yl)-1,3, 4-oxadiazole-2-thiol (97%; CAS No. 15264-63-8), sodium hydroxide (ACS reagent, ≥97%; CAS No. 1310-73-2), hydroxylamine hydrochloride (99.999%; CAS No. 5470-11 -1), terephthalic anhydride (ReagentPlus, 99%, CAS No. 623-27-8), 4-methylpyridine (99%; CAS No. 108-89-4), acetic anhydride (ReagentPlus, ≥99%; CAS No. 108- 24-7), dichloromethane (anhydrous, 99.8%; CAS No. 75-09-2), and methanol (HPLC, ≥99.9%; CAS No. 67-56-1) were purchased from Sigma Aldrich. Hydrochloric acid (trace metal, 34–37%; CAS No. 7647-01-0) was purchased from Fisher Scientific.

ζ -potential values were measured using a Malvern Zetasizer Nano-ZS with 1-ml curved capillary cuvettes (DTS1060). Optical absorption spectra were recorded on an Agilent 8453 and Perkin Elmer Lambda 650 spectrophotometer.

Scanning electron microscope (SEM) images were captured on a Hitachi S-4500.

785 nm Raman spectra were acquired on an Ocean Optics QE 6500.

The size of the nominal 140 nm gold colloidal particles was determined by sending the sample to EAG Laboratories for transmission electron microscopy (TEM) imaging. Images were analyzed using ImageJ software (https://imagej.nih.gov/ij/). A lot was considered nominally 140 nm if the number average particle size was within ± 9 nm of the nominal diameter, with the number average particle size determined by measuring 230 individual particles in TEM micrographs. The size-related characteristic chosen to describe individual particles was the diameter of the “equivalent circle” (CE), which corresponds to the diameter of a circle whose area is equal to the area of the orthogonal projection of the particle.

Diameter of gold nanoparticlesd (nm) for batches of nanoparticles with sizes 40, 60, 90 nm were calculated using the method described by Haiss et al. (Anal. Chem. 2007,79, 4215-4221), using the following equation:

where λ _spr denotes the position of the surface plasmon resonance peak in the extinction plot obtained from the colloid sample on a Perkin Elmer Lambda 650 operating in the UV/visible spectrum. A lot was considered nominally 40 nm, 60 nm, or 90 nm if the surface plasmon resonance peak position correlated with the diameter within ±9 nm of the nominal diameter.

Preparation of the initial solution of colloidal Au (S1 - S4)

Colloidal gold stock solutions (S1 - S4), characterized by Au nanoparticle size (nm), Au concentrations (mg/mL) and pH values shown in Table 1, were prepared as described below.

I.1 Preparation of 40 nm colloidal gold stock solution (S1)

In a jacketed and thoroughly cleaned 100 L glass reactor (ChemGlass), 79.5 L of 17 MΩ water were cooled to 3.5 ± 0.5 ^o C. Gold chloroaurate trihydrate (100 g) was added as 20 wt. % solution in water while stirring with a paddle mixer at a speed of 400 rpm. Tribasic sodium citrate dihydrate (174.3 g) was combined as 30 wt. % solution in water and hydroxylamine hydrochloride (155.6 g) in the form of 23.5 wt. % solution in water and added to the reactor. After 10 s, 800 μl of sodium borohydride was added to the reactor in the form of 0.063 wt. % solution in 0.01 N. sodium hydroxide. The reagents were allowed to react for 2 minutes and then poured into a clean drum to obtain a stock solution of 40 nm colloidal Au (S1) with a gold concentration of 0.25 mg Au/mL and a pH value of approximately 2.4.

I.2 Preparation of 60 nm colloidal gold stock solution (S2)

In a jacketed and thoroughly cleaned 100 L glass reactor (ChemGlass), 79.5 L of 17 MΩ water were cooled to 3.5 ± 0.5 ^o C. Gold chloroaurate trihydrate (200 g) was added as 20 wt. % solution in water while stirring with a paddle mixer at a speed of 400 rpm. Tribasic sodium citrate dihydrate (173.3 g) was combined as 30 wt. % solution in water and hydroxylamine hydrochloride (217.1 g) in the form of 17.5 wt. % solution in water and added to the reactor. After 10 s, 800 μl of sodium borohydride was added to the reactor in the form of 0.052 wt. % solution in 0.01 N. sodium hydroxide. The reagents were allowed to react for 2 minutes and then poured into a clean drum. The batch was diluted with 17 MΩ water to 160 L to obtain a stock solution of 60 nm colloidal Au (S2) with a gold concentration of 0.25 mg Au/mL and a pH of approximately 2.4.

I.3 Preparation of 90 nm colloidal gold stock solution (S3)

In a jacketed and thoroughly cleaned 100 L glass reactor (ChemGlass), 79.5 L of 17 MΩ water were cooled to 3.5 ± 0.5 ^o C. Gold chloroaurate trihydrate (200 g) was added as 20 wt. % solution in water while stirring with a paddle mixer at a speed of 400 rpm. Tribasic sodium citrate dihydrate (173.3 g) was combined as 30 wt. % solution in water and hydroxylamine hydrochloride (217.1 g) in the form of 17.5 wt. % solution in water and added to the reactor. After 10 s, 900 μl of sodium borohydride was added to the reactor in the form of 0.01 wt. % solution in 0.01 N. sodium hydroxide. The reagents were allowed to react for 2 minutes and then poured into a clean drum. The batch was diluted with 17 MΩ water to 160 L to obtain a stock solution of 90 nm colloidal Au (S3) with a gold concentration of 0.25 mg Au/mL and a pH of approximately 2.4.

I.4 Preparation of a stock solution of 140 nm colloidal gold (S4)

In a 2-liter glass bottle, 1.5 liters of water with a resistance of 17 MΩ were mixed at room temperature. Gold chloraurate trihydrate (2.5 g) was added as 20 wt. % solution in water with stirring. Tribasic sodium citrate dihydrate (15.37 g) was combined as 30.7 wt. % solution in water and hydroxylamine hydrochloride (8.37 g) in the form of 16.7 wt. % solution in water and added to the reactor. After 5 s, 25 μl of sodium borohydride was added to the reactor in the form of 0.01 wt. % solution in 0.01 N. sodium hydroxide. An additional stirring for 15 minutes yielded a stock solution of 140 nm colloidal Au (S4) with a gold concentration of 0.25 mg Au/mL and a pH of approximately 2.4.

Table 1. Results of studying the characteristics of initial solutions of colloidal Au (S1 - S4)

Preparation of the first colloid (A1 - A9) (step a) of the manufacturing method)

The first colloids (A1 - A9) were obtained from colloidal Au solutions S1, S2 and S4. If necessary (in the case of, for example, Au colloids A1 and A7), the stock solutions of colloidal Au were diluted with deionized water to obtain the Au concentration (mg/ml) indicated in Table 2. Additionally, if necessary (in the case of, for example, Au colloids A3, A4, A5, A6, A8, A9) the initial solutions of colloidal Au alone or after dilution were treated with an aqueous 0.1 mM NaOH solution or an aqueous 0.1 mM HCl solution in order to bring the pH value of the first colloids to the pH value , indicated in table 2.

Table 2. Results of studying the characteristics of the first colloids (A1 - A9)

Only stable first colloids, ie colloids in which there was no aggregation, were suitable for use in the production method of the present invention. The first colloids A1 - A9 were stable, i.e., the aggregation of Au nanoparticles in them could not be detected by visually detecting a color change from pink to purple. Moreover, as can be seen in FIG. 4, a number of Au colloids were stable and exhibited a zeta potential value that was less than or equal to -25 mV, as measured in Section IV below. Therefore, such Au colloids were also suitable for use as first colloids in the production method of the present invention.

Preparation of a second colloid containing Au nanoparticles and active Raman reporter molecules adsorbed on the surface of Au nanoparticles (D1 - D11) (step b) of the manufacturing method)

Au colloids (B1 - B8), characterized by Au concentrations (mg/ml) and pH values indicated in Table 3, were prepared from Au colloidal stock solutions S1 - S3. The preparation involved, if necessary (in the case of, for example, Au colloids B1 and B5), diluting the initial solutions of colloidal Au with deionized water so as to obtain the indicated concentration of Au (mg/ml), and adjusting the pH value by adding an aqueous 1 M NaOH solution in initial solutions of colloidal Au as is or after dilution.

Table 3. Results of studying the characteristics of Au B1 - B9 colloids

The active Raman reporter molecule 1,4-bis(( E )-2-(pyridin-4-yl)vinyl)benzene was synthesized as described below.

A 50 mL round bottom flask with a stirrer was charged with terephthalic anhydride (3.44 g, 25.7 mmol), 4-methylpyridine (9.57 g, 103 mmol) and acetic anhydride (25 mL). The mixture was refluxed until the aldehyde was completely removed (4 hours, checked by TLC). The reaction mixture was cooled to room temperature and quenched by pouring it into 100 ml of ice water. The cold mixture was neutralized to pH 7 with aqueous 6N. NaOH solution, and the resulting brown precipitate was filtered, washed with water and dried in air. Extraction with dichloromethane followed by concentration of the solvent to dryness provided a crude product, which was purified by silica gel flash column chromatography (methanol/dichloromethane: 5/95) to yield 0.875 g of the target active Raman reporter molecule as a yellow solid (12%).

The second colloid (D1 - D11), containing Au nanoparticles with active Raman reporter molecules adsorbed on their surface, was prepared by adding a certain volume of 0.1 mM solution of active Raman reporter molecules in ethanol to 20 ml of Au colloid (B1 - B5, B9 ) or 3 liters of Au colloid (B8), followed by stirring the resulting mixture for 30 minutes (in the case of the second colloid D1 - D7, D9 - D11)/1 hour (in the case of the second colloid D8) at room temperature. Table 4 provides a brief description of the active Raman reporter molecules and the volumes of the active Raman reporter molecule solution in ethanol that were used to prepare the second colloid D1 - D11.

Table 4. Preparation of second colloids D1 - D11

Treatment of 20 ml of Au colloids B6 and B7 with a pH value below the lowest pH value at which ( E )-1,2-di(pyridin-4-yl)ethene carried no net electrical charge, as predicted by Chemicalize.com, by 750 μl of a 0.1 mM solution of ( E )-1,2-di(pyridin-4-yl)ethene in ethanol followed by stirring the resulting mixture for 30 min at room temperature resulted in the formation of unstable colloids, as evidenced by visual detection of the change The color of the mixture changes from pink to purple when stirred. Unstable colloids could not be used in the manufacturing method of the present invention. To ensure the stability of the second colloid, i.e. to avoid aggregation of Au nanoparticles contained in the said colloid, it was important that during the production and storage of the said colloid, the ζ -potential value was less than or equal to -25 mV, and the pH value was above the most low pH value at which the active Raman reporter molecule adsorbed on Au nanoparticles carries no net electrical charge, and this value could be predicted, for example, using Chemicalize.com. In this sense, the pH value of the Au colloid (B1 - B5, B8, B9) used to prepare the second colloid was adjusted to a value that was higher than the lowest pH value at which the active Raman reporter molecule was treated with the active Raman reporter molecule before being treated with it. adsorbed on Au nanoparticles contained in the Au colloid (B1 - B5, B8, B9), does not carry a total electrical charge, and the indicated value could be predicted, for example, using Chemicalize.com.

Stability of the first and second colloids. In order to be used in the manufacturing method of the present invention, which produces SERS tags with a high number of small aggregates, the first and second colloids had to be stable, i.e., aggregation had to be avoided during the production and storage of said colloids nanoparticles This could be ensured by maintaining the ζ -potential value of these colloids at a level below or equal to -25 mV. As can be seen in FIG. 4 and Table 5, a number of Au colloids could be used as the first and second colloids in the method for making SERS tags of the present invention.

The ζ-potential of the first colloid A2 and the second colloids D2, D5 and D6 was measured as a function of pH at a concentration of 0.05 mg Au/mL at room temperature. The results are presented in Table 5 and graphically depicted in FIG. 4.

ζ-potential measurements were performed on a Malvern Zetasizer Nano-ZS with 1-ml curved capillary cuvettes. The physical properties of water at 25°C and gold were preloaded into 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 Au/mL.

The ζ-potential values of the first colloid A2 and the second colloid D2, D5 and D6 at approximately the pH used for the synthesis of the SERS tags of the present invention are presented in the 2nd column of Table 5. The ζ-potential values of the first colloid A2 and the second colloid D2, D5 and D6 at the pH at which colloids become unstable are presented in the 3rd column of Table 5. Colloid instability was determined by visually detecting a change in color of the colloid from pink to purple.

Table 5. Results of measuring the ζ-potential of the first and second colloids

V. Combining the first colloid and the second colloids, inducing aggregation and stopping aggregation (step c), d) and e) of the manufacturing method)

V.1 SERS tags containing Au nanoparticles of the same size (examples E1 - E8, E12 - E14)

V.1.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 the mixing tank using a magnetic stirrer. 30 seconds after combining the colloids, an aliquot of 125 μl was taken and diluted with water to 1 ml, thereby stopping aggregation. At this dilution (12.5 μg Au/ml, which corresponded to approximately 5.72*10 ⁹ Au nanoparticles/ml), aggregation ceased, and with the help of laser excitation at 785 nm and a QE65000 spectrometer purchased from Ocean Optics Inc., for the time required to accumulate charge by the LED in one second, the SERS signal was measured. The results of SERS signal measurements carried out on SERS tags manufactured as described above are shown in Table 6.

As presented in Examples E1 - E7 and E12 - E14 according to the method of the present invention as claimed herein and in Comparative Examples C1 - C3, selective aggregation of the first colloid particles and the second colloid particles did not occur at low pH values such as 1.8. and at pH values that were higher than the lowest pH at which active Raman reporter molecules had a net electrical charge of 0 to 0.3, as predicted by Chemicalize.com.

V.1.b Aggregation in a continuous flow system (example E8)

A schematic diagram of a continuous flow reactor used in the manufacturing method of the present invention is shown in FIG. 1b. The pressurized reservoir (111b) contained the second colloid, and the second pressurized reservoir (112b) contained the first colloid. To collect SERS tags, a vessel (114b) open to the atmosphere was used, which contained a concentrated polymer solution to stop the aggregation reaction. Clear fluorinated ethylene propylene (FEP) tubing from Cole Parmer® Scientific Experts with a nominal internal diameter of 8 mm was used for colloid transport. 30 cm of FEP tubing was used to connect the pressure reservoir (111b) to the T-connector (113b). 30 cm of FEP tubing was used to connect the pressure reservoir (112b) to the T-connector (113b). 7 m of FEP tubing was used to connect the T-connector (113b) to the collection vessel (114b). This section of tubing between the T-connector (113b) and the collection vessel (114b) was the section where aggregation occurred.

The colloids A8 (12 L) and D8 (3 L) described above were placed in reservoirs 112b and 111b, respectively. The reservoir (111b) containing the second colloid (D8) was pressurized so that the flow rate of the colloid was 0.66 L/min. The reservoir (112b) containing the first colloid (A8) was pressurized so that the flow rate of the colloid was 2.65 L/min. After combining in the T-connector, the combined colloid, characterized by a nanoparticle ratio of the first colloid A8 to the nanoparticles of the second colloid D8 of ≈4:1, had a pH value of approximately 4.8 and a flow rate in the aggregation tube of 3.31 L /min, which gave a linear speed of 1.1 m/s. SERS measurements were performed through a clear FEP tube at 1 m along the length of the aggregation tube using a probe connected by fiber optic cable to an Ocean Optics QE65000 spectrometer with excitation at 785 nm. The time for LED charge accumulation was reduced so that the measurement result at the mark at the end of the tube was equal to the value obtained from a 50 μl aliquot taken from the tube opening and diluted to 1 ml with water (12.5 μg Au/ml), i.e. value for a sample in which aggregation was stopped by dilution with water, and measured using laser excitation at 785 nm and an Ocean Optics QE65000 spectrometer with the LED charge storage time set to one second. The results of SERS measurements are presented graphically in Fig. 5 in the form of diamonds connected by a solid line. The SERS signal intensity from the end-of-tube sample measuring 12.5 μg Au/mL was 11,044 pps [±500].

V.2 The first colloid and the second colloid contained Au nanoparticles of different sizes (examples E9 - E11)

V.2.a Batch aggregation (examples 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 using a magnetic stirrer. The ratio of the number of nanoparticles of the first colloid to the number of nanoparticles of the second colloid was ≈6.3:1. After 30 seconds of stirring at a pH of approximately 2.9, aggregation was stopped by adding a polymer solution, and the resulting aggregates were further coated with silica according to the method described in US8497131B2 to obtain the target SERS tags. ~2-μL aliquots of SERS tags were dropped onto silica plates and dried. The sample was visualized on a Hitachi S-4500 field emission scanning electron microscope; the resulting image is shown in Fig. 3a. The SERS signal was measured using laser excitation at 785 nm and a QE65000 spectrometer purchased from Ocean Optics Inc., with the LED charge storage time set to one second. The SERS signal intensity measured for 12.5 μg Au/mL was 18,377 pps [±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 using a magnetic stirrer. The ratio of the number of nanoparticles of the first colloid to the number of nanoparticles of the second colloid was ≈2.65:1.

After 30 seconds of stirring at a pH of about 2.9, aggregation was stopped by adding a polymer solution, and the resulting aggregates were coated with silica according to the method described in US8497131B2 to obtain the target SERS tags. ~2-μL aliquots of SERS tags were dropped onto silica plates and dried. The sample was visualized on a Hitachi S-4500 field emission scanning electron microscope; the resulting image is shown in Fig. 3b.

V.2.b Aggregation in a flow system (example E11)

A schematic diagram of a continuous flow reactor used in the manufacturing method of the present invention is shown in FIG. 1b. The pressurized reservoir (111b) contained the second colloid, and the second pressurized reservoir (112b) contained the first colloid. An open vessel (collection vessel, 114b) was used to collect SERS tags and stop aggregation. Fluorinated ethylene propylene (FEP) tubing from Cole Parmer® Scientific Experts with a nominal internal diameter of 8 mm was used for colloid transport. 30 cm of clear FEP tubing was used to connect the pressure reservoir (111b) to the T-connector (113b). 30 cm of FEP tubing was used to connect the pressure reservoir (112b) to the T-connector (113b). 7 m of FEP tubing was used to connect the T-connector (113b) to the collection vessel (114b). This section of tubing between the T-connector (113b) and the collection vessel (114b) was the section where aggregation occurred.

The colloids A6 (12 L) and D8 (3 L) described above were placed in reservoirs 112b and 111b, respectively. The reservoir (111b) containing the second colloid (D8) was pressurized so that the flow rate of the colloid was 0.66 L/min. The reservoir (112b) containing the first colloid (A6) was pressurized so that the flow rate of the activated colloid was 2.65 L/min. After combining in the T-connector, the combined colloid, characterized by a nanoparticle ratio of the first colloid A6 to the nanoparticles of the second colloid D8 of ≈1.2:1, had a pH of approximately 4.8 and a flow rate in the aggregation tube of 3.31 l/min, which gave a linear speed of 1.1 m/s. SERS measurements were performed through a clear FEP tube at 1 m along the length of the aggregation tube using a probe connected by fiber optic cable to an Ocean Optics QE65000 spectrometer with excitation at 785 nm. The time for the LED to accumulate charge was reduced so that the measurement result at the mark on the end of the tube was equal to the value obtained from a 50 μl aliquot taken from the tube opening and diluted to 1 ml with water (12.5 μg Au/ml), (step e2 ), and measured using laser excitation at 785 nm and an Ocean Optics QE65000 spectrometer with the LED charge storage time set to one second. The results of these measurements are graphically presented in Fig. 5 in the form of circles connected by a dotted line. The SERS signal intensity from the end-of-tube sample measuring 12.5 μg Au/mL was 28,303 pps [±500].

Claims (30)

1. A method for producing tags for surface-enhanced Raman spectroscopy (SERS), comprising the following steps: a) obtaining a first colloid essentially consisting of nanoparticles with a plasmonic surface and essentially the same size, dispersed in an aqueous solvent, and a stabilizing agent adsorbed on the surface of said nanoparticles, and characterized by a ζ-potential value less than or equal to -25 mV; b) obtaining a second colloid essentially consisting of nanoparticles with a plasmonic surface and essentially the same size, dispersed in an aqueous solvent, active Raman reporter molecules adsorbed on the surface of said nanoparticles, and a stabilizing agent adsorbed on the surface of said nanoparticles, and characterized by a ζ value -potential less than or equal to -25 mV; c) combining the first colloid with the second colloid such that the ratio of the number of nanoparticles of the first colloid to the number of nanoparticles of the second colloid is from about 25:1 to about 1:1, preferably from about 5:1 to about 1:1, to obtain a third colloid; d) induction of nanoparticle aggregation using any of stages d1)–d3) or a combination thereof: d1) mixing the third colloid obtained in step c) at a pH ranging from about 2.2 to the lowest pH at which the active Raman reporter molecules have a net electrical charge of from 0 to 0.3; d2) adding a salt solution, preferably an inorganic salt solution, to the third colloid obtained in step c); d3) adding a water-soluble solvent to the third colloid obtained in step c); And e) stopping aggregation. 2. The method according to claim 1, characterized in that stage b) involves the following stages, carried out in the order b1)-b3): b1) obtaining a colloid essentially consisting of nanoparticles with a plasmonic surface and essentially the same size, dispersed in an aqueous solvent, and a stabilizing agent adsorbed on the surface of these nanoparticles, and characterized by a ζ-potential value 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 active Raman reporter molecules to be adsorbed to the surface of the nanoparticles carry no net electrical charge, while maintaining a ζ-potential value less than or equal to -25 mV, preferably below -40 mV; And b3) adding a solution of active Raman reporter molecules in a solvent to the colloid obtained in step b2), while maintaining the ζ-potential value below or equal to -25 mV. 3. The method according to claim 1 or 2, characterized in that the stabilizing agent is selected from carboxylic acids, salts of carboxylic acids, phosphoric acids, salts of phosphoric acids, ascorbic acid, salts of ascorbic acid and mixtures thereof. 4. Method according to any one of paragraphs. 1-3, characterized in that the nanoparticles of the second colloid have a submonolayer or monolayer of active Raman scattering reporter molecules absorbed on their surface. 5. Method according to any one of paragraphs. 2-4, characterized in that in step b3) the solution of active Raman reporter molecules in a solvent contains a mixture of two or more different active Raman reporter molecules. 6. Method according to any one of paragraphs. 1-5, characterized in that in step c) the ratio of the number of nanoparticles of the first colloid to the number of nanoparticles of the second colloid is from about 4:1 to about 3:1. 7. Method according to any one of paragraphs. 1-6, characterized in that step d1) further involves adding an acidic solution to the third colloid obtained in step c) with stirring, so that the pH value of the resulting colloid is in the range from about 2.2 to the lowest pH value, in which active Raman reporter molecules have a net electrical charge of 0 to 0.3. 8. Method according to any one of paragraphs. 1-6, characterized in that the pH of the first colloid is adjusted such that the pH of the third colloid obtained in step d1) is from about 2.2 to the lowest pH value at which the active Raman reporter molecules have a net electrical charge of 0 up to 0.3. 9. Method according to any one of paragraphs. 1-8, characterized in that stage e) includes any of the following stages e1)–e4): e1) adjusting the pH of the colloid obtained in step d) to a value greater than the lowest pH value at which the active Raman reporter molecules that are to be adsorbed on the surface of the nanoparticles carry no net electrical charge; e2) diluting the colloid obtained in step d) with water; e3) adding the polymer to the colloid obtained in step d); e4) adding a dielectric material precursor to the colloid obtained in step d). 10. Method according to any one of paragraphs. 1-9, characterized in that step e) actually consists of step e3), and the manufacturing method additionally involves diluting the colloid obtained in step e3) with water and/or coating the SERS tags with a dielectric material. 11. Method according to any one of paragraphs. 1-10, characterized in that stages c) and d) are carried out simultaneously in a continuous flow system. 12. The method according to claim 11, characterized in that step e) is carried out in a continuous flow system. 13. Method according to any one of paragraphs. 1-12, characterized in that the nanoparticles of the first colloid and the nanoparticles of the second colloid have the same size. 14. Method according to any one of paragraphs. 1-12, characterized in that the size of the nanoparticles of the first colloid differs from the size of the nanoparticles of the second colloid. 15. Method according to any one of paragraphs. 1-14, characterized in that the plasmonic surface of nanoparticles in the first colloid and/or the plasmonic surface of nanoparticles in the second colloid are made of gold.