Method for manufacturing surface-enhanced Raman spectroscopy labels

Abstract

The present invention relates to the field of methods for producing surface-enhanced Raman spectroscopy (SERS) tags. The methods according to the present invention are reproducible and versatile and enable the convenient production of large quantities of SERS tags characterized by a narrow size distribution and a high proportion of low-number aggregates. The SERS tags produced by the methods of the present invention described herein provide an increased overall SERS response.

Description

Technical Field

[0001] The present invention relates to the field of methods for producing surface-enhanced Raman spectroscopy (SERS) tags. The methods according to the present invention are reproducible and versatile and enable the convenient production of large quantities of SERS tags characterized by a narrow size distribution and a high proportion of low-number aggregates. The SERS tags produced by the methods of the present invention described herein provide an increased overall SERS response. Background Art

[0002] Surface enhanced Raman spectroscopy (SERS) tags have proven useful in a variety of applications, including product labeling for identification and authentication purposes, as well as high-throughput multiplex screening in microarray technology, diagnostics, and bioimaging. SERS tags are aggregates of nanoparticles that have a plasmonic surface and have Raman-active reporter molecules adsorbed on their surface. The nanoparticles that have a plasmonic surface are responsible for generating the electric field required for Raman amplification, while the Raman-active reporter molecules provide the unique vibrational fingerprint of the SERS tag. Typically, the aggregates are coated with an outer coating that a) isolates the SERS tag from the external medium, thereby preventing the Raman-active reporter molecules from leaching out of the SERS tag and protecting the SERS tag from contamination by the external medium that may cause vibrational noise, b) increases the colloidal stability of the SERS tag, and c) provides a surface that is convenient for further chemical functionalization. To date, polymers and silica have been used as outer coatings.

[0003] Due to the strong dependence of plasmonic properties on the aggregation state of nanoparticles, the fabrication of SERS tags with a large number of low-number aggregates is highly desirable to obtain an increased overall SERS response. SERS tags with a large number of low-number aggregates are fabricated by post-synthesis sorting techniques or by controlled aggregation synthesis methods.

[0004] For example, applying field flow fractionation to a mixture containing single nanoparticles and nanoparticle aggregates ranging from dimers to octamers resulted in fractions enriched in dimer (10%), trimer (21%), and tetramer (13%) SERS tags, but which contained a high percentage of single nanoparticles (52%) (J. Am. Chem. Soc. 2010, 132, 10903-10910). By using post-synthesis sorting in a high-viscosity density gradient medium (such as an aqueous iodixanol density gradient medium), a mixture containing single nanoparticles and aggregates ranging from dimers to dodecamers was enriched in dimer (32%) and trimer (32%) SERS tags (US9802818B2). In addition to being time-consuming and expensive, post-synthesis sorting techniques require the use of harsh conditions (e.g., high viscosity reagents) that render uncoated SERS tags unstable. Therefore, post-synthesis sorting techniques are only compatible with silica- or polymer-coated SERS tags. Therefore, controlled aggregation synthesis methods have great advantages for fabricating SERS tags with narrow size distribution and a high proportion of low number of aggregates.

[0005] Solid support-assisted aggregation allows the production of SERS tags with a narrow size distribution. Ruan et al. (Adv. Optical Mater. 2014, 2, 65-73) describe the synthesis of an asymmetric core-satellite SERS tag with 18 ± 2 Au nanoparticle satellites with an average diameter of 24 nm, each Au nanoparticle core having an average diameter of 180 nm. The synthesis involves adsorbing Au nanoparticle cores stabilized by cetyltrimethylammonium bromide onto a glass slide or silicon wafer coated with indium tin oxide. The functionalized solid support is then immersed in a water / acetonitrile solution of 4-aminothiophenol to adsorb Raman-active reporter molecules on the surface of the Au nanoparticles, followed by immersion in a suspension of Au nanoparticles with an average diameter of 24 nm for 1 hour. The aggregation state of the SERS tag can be controlled by controlling the immersion time of the functionalized solid support in the suspension of Au nanoparticle satellites and / or reducing the concentration of the Au nanoparticle satellites in the suspension. The method described by Ruan et al. relies on the use of hexadecyltrimethylammonium bromide surfactant to stabilize the Au colloids, which significantly reduces the surface available for adsorption of Raman-active reporter molecules onto the Au nanoparticle cores, thereby reducing the intensity of the SERS signal provided by the SERS tag. In order to tether the Au nanoparticle satellites to the Au nanoparticle cores, the aggregation method uses Raman-active reporter molecules that have two functional groups with affinity for the Au surface. Therefore, this method is only suitable for the manufacture of SERS tags with Raman-active reporter molecules that have two functional groups with affinity for the Au surface, which represents a high limitation in terms of Raman-activated reporter molecules used as SERS tag fingerprints. In addition, the method involves long reaction times and is not suitable for the convenient manufacture of large quantities of SERS tags.

[0006] Yoon et al. (ACS Nano 2012, 8, 7199-7208) described another solid support-assisted assembly method for SERS tags with narrow size distribution. The method is based on the size-dependent desorption tendency of Au nanoparticles adsorbed on an amino-functionalized glass slide and the use of alkanedithiol to tether the Au nanoparticle satellites to the Au nanoparticle core. A SERS tag was produced with 13±3 nanoparticle satellites with an average diameter of 13 nm, each with an average diameter of 51 nm and an average diameter of 13 nm. The method developed by Yoon et al. appears to be able to incorporate any Raman-active reporter molecule into the SERS tag. However, the method is limited in terms of the size of Au nanoparticles that can be used and the surface on the Au nanoparticle core that can access the Raman-active reporter molecule. In addition, the method requires a long reaction time and is not suitable for the convenient production of large quantities of SERS tags.

[0007] Therefore, there is still a need for a reproducible, cost-effective and universal method for fabricating SERS tags that can produce a large number of SERS tags characterized by a narrow size distribution and a high proportion of low number aggregates in a convenient manner. SERS tags with a large number of low number aggregates are very desirable for obtaining an increased overall SERS response. Summary of the Invention

[0008] Therefore, the object of the present invention is to provide a general, cost-effective and reproducible method for producing SERS tags, which can produce a large number of SERS tags in a convenient manner, characterized by a narrow size distribution and a high proportion of low number of aggregates. This is achieved by a method for producing a surface enhanced Raman spectroscopy (SERS) tag, preferably a SERS tag for use as a security element, comprising the following steps:

[0009] a) providing a first colloid, the first colloid consisting essentially of nanoparticles having a plasmonic surface and substantially the same size dispersed in an aqueous solvent, and a stabilizer adsorbed on the surfaces of the nanoparticles, and the first colloid having a zeta potential value lower than or equal to -25 mV;

[0010] b) providing a second colloid consisting essentially of nanoparticles having a plasmonic surface and substantially the same size dispersed in an aqueous solvent, a Raman-active reporter molecule adsorbed on the surface of the nanoparticles, and a stabilizer adsorbed on the surface of the nanoparticles, and having a zeta potential value less than or equal to -25 mV;

[0011] c) combining the first colloid and the second colloid such that the ratio of the number of the nanoparticles of the first colloid to the number of the nanoparticles of the second colloid is between about 25:1 and about 1:1, preferably about 5:1 and about 1:1, more preferably 4:1 and about 3:1, to provide a third colloid;

[0012] d) inducing aggregation of nanoparticles by any one of steps d1) to d3) or a combination thereof:

[0013] d1) mixing the third colloid obtained in step c) at a pH between about 2.2 and the lowest pH value at which the net charge of the Raman-active reporter molecule is between 0 and 0.3;

[0014] d2) adding a salt solution, preferably an inorganic salt solution, to the third colloid obtained in step c);

[0015] d3) adding a water-miscible solvent to the third colloid obtained in step c); and

[0016] e) Stop gathering.

[0017] Preferably, step b) of the method claimed and described herein comprises the following steps performed in the order b1) to b3):

[0018] b1) providing a colloid consisting essentially of nanoparticles having a plasmonic surface and substantially the same size dispersed in an aqueous solvent, and a stabilizer adsorbed on the surface of the nanoparticles, wherein the colloid has a zeta potential value lower than or equal to −25 mV;

[0019] b2) adjusting the pH of the colloid to the lowest pH value at which the Raman-active reporter molecules adsorbed on the surface of the nanoparticles do not carry a net charge, while maintaining the zeta potential value below or equal to -25 mV, preferably below -40 mV; and

[0020] b3) adding a solution of the Raman-active reporter molecule in a solvent to the colloid obtained in step b2) while maintaining the zeta potential value lower than or equal to -25 mV.

[0021] In a further preferred embodiment, steps c) and d) are performed simultaneously in a continuous flow system.In a further preferred embodiment, the nanoparticle size of the first colloid is different from the nanoparticle size of the second colloid.

[0022] As shown in Examples E1-E14, the methods claimed herein allow for the integration of various Raman-active reporter molecules into SERS tags and are not limited to combinations of nanoparticles of a particular size, but also enable the synthesis of SERS tags comprising nanoparticles having the same size as demonstrated, for example, in Examples E1-E8 and E12-E14. Further, the manufacturing methods claimed and described herein provide for SERS tags having, for example, Figure 3a and Figure 3b The narrow size distribution shown and e.g. Figure 3a and Figure 3b A high proportion of SERS tags with low numbers of aggregates is shown, and the reaction time is significantly shorter than methods known in the literature. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1aA method for manufacturing a SERS tag according to the present invention is schematically shown. The method comprises combining a second colloid (120a) consisting essentially of nanoparticles (121a) having a plasmonic surface and substantially the same size dispersed in an aqueous solvent (not shown), wherein the nanoparticles have adsorbed Raman-active reporter molecules (122a) and a stabilizer (not shown) on their surfaces, with a first colloid (130a) consisting essentially of nanoparticles having a plasmonic surface and substantially the same size dispersed in an aqueous solvent (not shown), wherein the nanoparticles have adsorbed a stabilizer (not shown) on their surfaces, and inducing aggregation of the nanoparticles to provide a SERS tag (140a).

[0024] Figure 1b An embodiment of a method for manufacturing a SERS tag according to the present invention is schematically shown. In this specific manufacturing method, a continuous flow reactor comprises two pressurized tanks (111b, 112b), a tee (113b), and a collection container (114b), wherein the two pressurized tanks (111b, 112b) are each connected to the tee (113b) via a pipe, and the tee (113b) itself is connected to the collection container (114b) via a pipe. The manufacturing method includes simultaneously combining and mixing a first colloid (130b) and a second colloid (120b) in a pipeline connecting a three-way connector (113b) to a collection container (114b) of a continuous flow reactor at a pH between about 2.2 and the lowest pH value at which the net charge of the Raman-active reporter molecule is between 0 and 0.3, wherein the first colloid (130b) is provided by a pressurized tank (112b) and is essentially composed of nanoparticles with a plasmonic surface and essentially the same size dispersed in an aqueous solvent (not shown), wherein the nanoparticles have a stabilizer (not shown) adsorbed on their surface, and the second colloid (120b) is provided by a pressurized tank (111b) and is essentially composed of nanoparticles with a plasmonic surface and essentially the same size dispersed in an aqueous solvent (not shown), wherein the nanoparticles have a Raman-active reporter molecule (122b) and a stabilizer (not shown) adsorbed on their surface. Mixing at a pH between about 2.2 and the lowest pH where the net charge of the Raman-active reporter molecule is between 0 and 0.3 induces nanoparticle aggregation.The aggregation step is stopped in a collection vessel (114b), thereby providing a target SERS tag (140b).

[0025] Figure 1cAn embodiment of a method for manufacturing a SERS tag according to the present invention is schematically shown. In this specific manufacturing method, a continuous flow reactor comprises three pressurized tanks (111c, 112c, 150c), a tee (113c), and a collection container (114c). The two pressurized tanks (111c, 112c) are each connected to the tee (113c) via a pipe, and the tee (113c) itself is connected to the collection container (114c) via a pipe. The manufacturing method includes simultaneously combining and mixing a first colloid (130c) and a second colloid (120c) in a pipeline connecting a three-way connector (113c) to a collection container (114c) of a continuous flow reactor at a pH between about 2.2 and the lowest pH value at which the net charge of the Raman-active reporter molecule is between 0 and 0.3, wherein the first colloid (130c) is provided by a pressurized tank (112c) and is essentially composed of nanoparticles with a plasmonic surface and essentially the same size dispersed in an aqueous solvent (not shown), wherein the nanoparticles have a stabilizer (not shown) adsorbed on their surface, and the second colloid (120c) is provided by a pressurized tank (111c) and is essentially composed of nanoparticles with a plasmonic surface and essentially the same size dispersed in an aqueous solvent (not shown), wherein the nanoparticles have a Raman-active reporter molecule (122c) and a stabilizer (not shown) adsorbed on their surface. Mixing induces nanoparticle aggregation at a pH between about 2.2 and the lowest pH value at which the net charge of the Raman-active reporter molecule is between 0 and 0.3. The aggregation step is stopped by introducing water stored in a pressurized tank (150c) into a pipe leading to a collection container (114c) to dilute the colloid, introducing an alkaline solution stored in a pressurized tank (150c) into a pipe leading to a collection container (114c), introducing a polymer stored in a pressurized tank (150c) into a pipe leading to a collection container (114c), or introducing a dielectric material precursor stored in a pressurized tank (150c) into a pipe leading to a collection container (114c), thereby providing a target SERS tag (140c).

[0026] Figure 2a -c shows the change of SERS signal intensity with aggregation reaction time: Figure 2a shows the change of SERS intensity of the SERS tag manufactured as described in Example E1 as a function of aggregation reaction time; Figure 2b shows the variation of the SERS intensity of the SERS tag manufactured as described in Example E14 with the aggregation reaction time; Figure 2cFigure 1 shows the SERS intensity of a SERS tag fabricated as described in Example E13 as a function of the aggregation reaction time. The horizontal axis corresponds to the aggregation reaction time in seconds, and the vertical axis corresponds to the SERS response to 785 nm excitation. The reported SERS intensity was measured at a given gold concentration with an integration time of 100 ms per second, and the signal intensity is associated with a sample diluted to 12.5 μg Au / mL and scanned with a 1-second integration time.

[0027] Figure 3a -b shows the results of Example E9 ( Figure 3a ) and 10( Figure 3b ) SEM images of SERS tags containing nanoparticles of different sizes manufactured by the present invention. As confirmed by the SEM images, the SERS tags manufactured according to the present invention are rich in low-number aggregates, such as dimers, trimers and tetramers, and exhibit a narrow size distribution.

[0028] Figure 4 Graph illustrating the zeta potential values ​​of the first colloid A2 (drawn as solid circles) and the second colloids D2 (drawn as solid triangles), D5 (drawn as solid diamonds), and D6 (drawn as solid squares) when the colloids are titrated with a 1N HCl aqueous solution or a 1N NaOH aqueous solution. The horizontal axis corresponds to the pH value and the vertical axis corresponds to the zeta potential value (mV). The conditions under which aggregation is observed (i.e., the colloid is unstable) are circled. Figure 4 It was demonstrated that various colloids characterized by a zeta potential value below -25 mV are stable. In the production method according to the invention, such colloids can be used as the first colloid and the second colloid, respectively.

[0029] Figure 5 Figure 2 shows the evolution of the SERS signal intensity for colloids aggregated in a flow system. Each measurement was taken at the 1-meter mark along a 7-meter long transparent FEP tube. The diamonds connected by solid lines represent data taken from a SERS tag manufactured as described in Example E8. The circles connected by dashed lines represent data taken from a SERS tag manufactured as described in Example E11. DETAILED DESCRIPTION

[0030] definition

[0031] The following definitions are intended to clarify the meaning of terms discussed in the specification and recited in the claims.

[0032] As used herein, the indefinite article "a" or "an" refers to one or more than one and does not necessarily limit the noun it designates to a singular.

[0033] As used herein, the term "at least" is intended to define one or more than one, eg, one or two or three.

[0034] As used herein, the term "comprising" is intended to be non-exclusive and open-ended. Thus, for example, a solution comprising compound A may include other compounds in addition to A. However, the term "comprising" also encompasses the more restrictive meanings of "consisting essentially of" and "consisting of" as specific embodiments thereof, such that, for example, a "solution comprising A, B, and optionally C" may also consist (essentially) of A and B or (essentially) of A, B, and C.

[0035] Insofar as the present description refers to “preferred” embodiments / features, combinations of these “preferred” embodiments / features should also be considered disclosed, as long as this combination of “preferred” embodiments / features makes technical sense.

[0036] As used herein, the term "about" means that the amount or value in question can be a specified certain value or some other value in the vicinity thereof. Typically, the term "about" indicating a particular value is intended to indicate a range within ±5% of that value. As an example, the phrase "about 100" indicates a range of 100 ± 5, i.e., a range from 95 to 105. Preferably, the range indicated by the term "about" indicates a range within ±3%, more preferably ±1%, of that value. Typically, when the term "about" is used, it is expected that similar results or effects according to the present invention can be obtained within a range of ±5% of the specified value.

[0037] Surprisingly, it has been found that a method for producing a surface enhanced Raman spectroscopy (SERS) tag, preferably a SERS tag for use as a security element, comprises the following steps:

[0038] a) providing a first colloid, the first colloid consisting essentially of nanoparticles having a plasmonic surface and substantially the same size dispersed in an aqueous solvent, and a stabilizer adsorbed on the surfaces of the nanoparticles, and the first colloid having a zeta potential value lower than or equal to -25 mV;

[0039] b) providing a second colloid consisting essentially of nanoparticles having a plasmonic surface and substantially the same size dispersed in an aqueous solvent, a Raman-active reporter molecule adsorbed on the surface of the nanoparticles, and a stabilizer adsorbed on the surface of the nanoparticles, and having a zeta potential value less than or equal to -25 mV;

[0040] c) combining the first colloid and the second colloid such that the ratio of the number of the nanoparticles of the first colloid to the number of the nanoparticles of the second colloid is between about 25:1 and about 1:1, preferably about 5:1 and about 1:1, more preferably about 4:1 and about 3:1, to provide a third colloid;

[0041] d) inducing aggregation of nanoparticles by any one of steps d1) to d3) or a combination thereof:

[0042] d1) mixing the third colloid obtained in step c) at a pH between about 2.2 and the lowest pH value at which the net charge of the Raman-active reporter molecule is between 0 and 0.3;

[0043] d2) adding a salt solution, preferably an inorganic salt solution, to the third colloid obtained in step c);

[0044] d3) adding a water-miscible solvent to the third colloid obtained in step c); and

[0045] e) Stop gathering;

[0046] This method provides a large number of SERS tags with a narrow size distribution and a high proportion of low-number aggregates in a cost-effective and convenient manner. This method does not have any restrictions on the size of the Raman-active reporter molecules used as the fingerprint of the SERS tag or the nanoparticles with plasmonic surfaces contained in the first and second colloids, thereby enabling the production of a variety of SERS tags.

[0047] As known to those skilled in the art and used herein, a SERS tag comprises an aggregate of nanoparticles present on a plasmon surface and Raman-active reporter molecules adsorbed on the nanoparticle surface. The nanoparticles present on the plasmon surface are responsible for generating the electric field required for Raman amplification, while the Raman-active reporter molecules provide the unique vibrational fingerprint of the SERS tag. The SERS tag may further include an outer coating that isolates the nanoparticle aggregate of the Raman-active molecules adsorbed on the surface from the external medium. Thus, the outer coating a) isolates the SERS tag from the external medium, thereby preventing the Raman-active reporter molecules from leaching from the SERS tag and protecting the SERS tag from contamination by the external medium that may cause spurious peaks, b) increases the colloidal stability of the SERS tag, and c) provides a surface that is convenient for further chemical functionalization. The outer coating includes silica and polymers such as poly(ethyleneimine) (PEI), poly(styrene-alt-maleic acid) sodium salt (PSMA), and poly(diallyldimethylammonium chloride) (PDADMAC).

[0048] Due to the SERS signal exhibited, the SERS tags obtained by the manufacturing methods claimed and described herein are particularly useful as security elements for protecting documents and articles from forgery and illegal copying. As used herein, the term "security element" refers to an element that can be incorporated into or applied to a security document or article for the purpose of determining its authenticity and protecting it from forgery and illegal copying. The security element can be a mark, image, pattern or graphic element printed, coated or sprayed onto the security element or article using ink, varnish or coating composition, wherein the ink, varnish or coating composition contains the SERS tag obtained by the manufacturing method according to the present invention. Optionally, when integrated into the substrate of a security document, the SERS tag can be used as a security element. The terms "security document" and "security article" refer to a document or article that has a value that makes it potentially susceptible to attempts to forge or illegally copy, and is generally protected from forgery or fraud by at least one security feature. As used herein, the term "security article" covers all articles that should be protected from forgery and / or illegal copying in order to guarantee their contents. Examples of security documents include, but are not limited to, valuable documents and valuable commercial goods. Typical examples of valuable documents include, but are not limited to, banknotes, contracts, bills, checks, vouchers, tax stamps and tax labels, agreements, and identification documents such as passports, ID cards, visas, bank cards, credit cards, transaction cards, passes, and admission tickets. Valuable commercial goods include packaging materials, particularly for cosmetics, functional foods, pharmaceuticals, alcoholic beverages, tobacco products, beverages or foods, electrical / electronic products, textiles, or jewelry, i.e., packaging materials that should be protected from counterfeiting and / or illegal copying to ensure the authenticity of the packaged contents, such as genuine pharmaceuticals. Examples of packaging materials include, but are not limited to, labels such as identification brand labels, tamper-evident labels, and seals.

[0049] Within the meaning of the present invention, the term "nanoparticle" is defined as a single particle having a size corresponding to a maximum physical dimension (e.g., length, diameter, etc.) in the range of 20±5 nm to 160±5 nm, preferably in the range of 40±5 nm to 140±5 nm. The nanoparticles used in the present invention have a plasmonic surface, i.e., the nanoparticles have an outer surface that can enhance Raman scattering of Raman-active molecules. The outer surface of the nanoparticle 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 nanoparticles used in the present invention can be solid or hollow, and are preferably solid. Solid nanoparticles can be made of a single material (i.e., the SERS-enhancing material of the outer surface of the nanoparticle) or made of more materials (i.e., one or more materials of the core of the nanoparticle can be different from the SERS-enhancing material of the outer surface of the nanoparticle). Hollow nanoparticles are nanoparticles whose core is a void space. The nanoparticles can have any shape that can be manufactured. Preferably, the nanoparticles are solid Au nanoparticles. Preferably, the nanoparticles have a shape selected from the group consisting of a sphere, a spheroid, a rod, a disk, a prism, and a cube, more preferably have a shape selected from a sphere and a spheroid, and even more preferably the nanoparticles have a spheroid shape.

[0050] As used herein, the term "nanoparticles having substantially the same size" means that the nanoparticles have a size corresponding to a maximum physical dimension (e.g., length, diameter, etc.) that is within ±20 nm, preferably ±10 nm, of the average size of the nanoparticles as determined by electron microscopy, such as transmission electron microscopy (TEM) or scanning electron microscopy (SEM), or, where applicable, by the method of Haiss et al. (Anal. Chem. 2007, 79, 4215-4221).

[0051] The nanoparticles of the first colloid, the second colloid, and the nanoparticles of the colloid provided in step b1) are dispersed in an aqueous solvent. As used herein, the term "aqueous solvent" refers to water and a mixture of water and one or more water-miscible solvents, wherein the water-miscible solvent is preferably selected from the list consisting of methanol, ethanol, propanol, isopropanol, tetrahydrofuran, N-methyl-2-pyrrolidone (NMP), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), acetone, and acetonitrile.

[0052] The first colloid provided in step a) of the manufacturing method, the second colloid provided in step b) of the manufacturing method, and the colloid provided in step b1) of the manufacturing method do not contain a polymer or surfactant for stabilization purposes. Colloid stabilization using polymers (e.g., polyvinyl pyrrolidone) or surfactants (e.g., hexadecyltrimethylammonium bromide) is well known in the art. However, since polymers and surfactants are added to the colloids before adding the Raman-active reporter molecules, they significantly reduce the surface available for adsorption of Raman-active reporter molecules on the nanoparticles and increase the spacing between the colloidal particles within the aggregate, resulting in the SERS tag exhibiting a lower SERS signal intensity. In order to overcome this shortcoming, the first colloid, the second colloid, and the colloid provided in step b1) are surfactant-free and polymer-free.

[0053] The stabilizer adsorbed on the surface of the nanoparticles of the first colloid, the second colloid, and the colloid provided in step b1) is preferably selected from carboxylic acids, carboxylates, phosphoric acid, phosphates, ascorbic acid, ascorbates, and mixtures thereof. In order to avoid competition between the Raman-active reporter molecule and the stabilizer during adsorption of the Raman-active reporter molecule on the nanoparticle surface, the stabilizer preferably does not contain a group that exhibits affinity for the SERS enhancing material (particularly gold). Examples of such groups are nitrogen-containing groups, sulfur-containing groups, acetylene groups, cyano groups, and isonitrile groups.

[0054] As used herein, the term "carboxylic acid" refers to an organic compound containing a carboxyl group (C(═O)OH), and encompasses monocarboxylic acids (i.e., organic compounds containing a single carboxyl group) such as lactic acid, as well as polycarboxylic acids (i.e., organic compounds containing two or more carboxyl groups) such as citric acid.

[0055] As used herein, the term "carboxylate" refers to the sodium or potassium salt of a carboxylic acid.

[0056] Preferably, the stabilizer is selected from carboxylic acids, carboxylates, ascorbic acid, ascorbates, and mixtures thereof. Even more preferably, the stabilizer is selected from citric acid, citrates, lactic acid, lactates, ascorbic acid, ascorbates, and mixtures thereof. Citrates include monosodium dihydrogen citrate, disodium hydrogen citrate, trisodium citrate, monopotassium dihydrogen citrate, dipotassium hydrogen citrate, and tripotassium citrate. Lactates include sodium lactate and potassium lactate. Ascorbates include sodium ascorbate and potassium ascorbate. In a most preferred embodiment, the stabilizer is selected from citric acid, monosodium dihydrogen citrate, disodium hydrogen citrate, trisodium citrate, monopotassium hydrogen citrate, dipotassium hydrogen citrate, and tripotassium citrate, and mixtures thereof.

[0057] Advantageously, the fabrication methods claimed and described herein enable the integration of any Raman-active reporter molecule into a SERS tag. Preferred Raman-active reporter molecules include fully conjugated molecules comprising an aryl group substituted with one or more substituents selected from the group consisting of NR 1 R 2 , -SH, -≡, ≡N and -N═, preferably -NR 1 R 2 and -SH, and / or N-containing heteroaryl and / or S-containing heteroaryl, wherein the residue R 1 and R 2 are independently selected from -H and alkyl, preferably -H and C1-C4 alkyl.

[0058] As known to those skilled in the art of organic chemistry, a fully conjugated molecule is a molecule having a conjugated electron system extending over the entire molecule. A conjugated electron system is a system of connected p orbitals with delocalized electrons.

[0059] As is well known to those skilled in the art, an "aryl group" is a group derived from a monocyclic or polycyclic aromatic hydrocarbon compound by removing a hydrogen atom from a ring carbon atom. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthrenyl, and pyrenyl.

[0060] S-containing aromatic heterocyclic compounds are aromatic compounds containing a sulfur heteroatom as part of a cyclic conjugated π system. As part of the cyclic conjugated π system, the S-containing aromatic heterocyclic compound may further contain one or more nitrogen atoms. Examples of S-containing heteroaryl groups include, but are not limited to, thienyl, thiazolyl, isothiazolyl, thiadiazolyl, benzothienyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, imidazothiazolyl, and imidazothiadiazolyl.

[0061] N-containing aromatic heterocyclic compounds are aromatic compounds comprising at least one nitrogen heteroatom as part of a cyclic conjugated π system. As part of a cyclic conjugated π system, N-containing aromatic heterocyclic compounds may further comprise one or more oxygen atoms. Examples of N-containing heteroaryl groups include, but are not limited to, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, benzimidazolyl, indazolyl, benzotriazolyl, pyridyl, pyrimidinyl, pyridazinyl, triazinyl, quinolyl, isoquinolyl, naphthyl, quinolizinyl, cinnolinyl, phthalazinyl, quinoxalinyl, purinyl, azaphenanthrenyl, diazaphenanthrenyl, azaanthryl, diazaanthryl, azapyrenyl, diazapyrenyl, oxazolyl, isoxazolyl, oxadiazolyl, benzoxazolyl, benzisoxazolyl, and benzoxadiazolyl.

[0062] Preferred Raman-active reporter molecules include, but are not limited to:

[0063] - a completely conjugated compound consisting of an aryl group substituted with one or more substituents selected from -NR 1 R 2 , -SH, -≡, -≡N and -N═, preferably -NR 1 R 2 and -SH, the fully conjugated compound is directly or via a linker group -L 1 - is attached to an aryl group substituted by one or more substituents selected from the list consisting of amino (-NH2), N-alkylamino, N,N-dialkyl-amino, thiol, ethynyl, cyano and isonitrile groups, N-containing heteroaryl groups, or S-containing heteroaryl groups, wherein

[0064] Substituent R 1 and R 2 has the meaning defined herein;

[0065] Linker-L 1 -Selected from-CR 8 ═CR 9 -、-N=N-、-≡-、-CR 10 ═CR 11 -o-C6H4-、-CR 10 ═CR 11 -m-C6H4-、-CR 10 ═CR 11 -p-C6H4-、-CR 10 ═CR 11 -o-C6H4-CR 12 ═CR 13 -、-CR 10 ═CR 11 -m-C6H4-CR 12 ═CR 13 -、-CR 10 ═CR 11 -p-C6H4-CR 12 ═CR 13 -、-CR 14 ═NN═CR 15 -、

[0066]

[0067]

[0068] and

[0069] Substituent R 8 –R 15 is selected from hydrogen, alkyl, alkoxy, alkylthio, formyl, cyano, nitro, halide, hydroxycarbonyl and alkoxycarbonyl;

[0070] - a completely conjugated compound consisting of a N-containing heteroaryl group, wherein the completely conjugated compound is directly or via a linker group -L 1 - is linked to an N-containing heteroaryl group or an S-containing heteroaryl group,

[0071] in

[0072] Linker-L 1 -Selected from-CR 8 ═CR 9 -、-N=N-、-≡-、-CR 10 ═CR 11 -o-C6H4-、-CR 10 ═CR 11 -m-C6H4-、-CR 10 ═CR 11 -p-C6H4-、-CR 10 ═CR 11 -o-C6H4-CR 12 ═CR 13 -、-CR 10 ═CR 11 -m-C6H4-CR 12 ═CR 13 -、-CR 10 ═CR 11 -p-C6H4-CR 12 ═CR 13 -、-CR 14 ═NN═CR 15 -、

[0073]

[0074]

[0075] and

[0076] Substituent R 8 –R 15 is selected from hydrogen, alkyl, alkoxy, alkylthio, formyl, cyano, nitro, halide, hydroxycarbonyl and alkoxycarbonyl;

[0077] - a completely conjugated compound consisting of a S-containing heteroaryl group, wherein the completely conjugated compound is directly or via a linker group -L 1 - is connected to a S-containing heteroaryl group,

[0078] in

[0079] Linker-L 1 -Selected from-CR 8 ═CR 9-、-N=N-、-≡-、-CR 10 ═CR 11 -o-C6H4-、-CR 10 ═CR 11 -m-C6H4-、-CR 10 ═CR 11 -p-C6H4-、-CR 10 ═CR 11 -o-C6H4-CR 12 ═CR 13 -、-CR 10 ═CR 11 -m-C6H4-CR 12 ═CR 13 -、-CR 10 ═CR 11 -p-C6H4-CR 12 ═CR 13 -、-CR 14 ═NN═CR 15 -、

[0080]

[0081] And the substituent R 8 –R 15 is selected from hydrogen, alkyl, alkoxy, alkylthio, formyl, cyano, nitro, halide, hydroxycarbonyl and alkoxycarbonyl;

[0082] and

[0083] - a fully conjugated compound consisting of an aromatic group substituted with one or more, preferably at least two, substituents selected from -NR 1 R 2 , -SH, -≡, -≡N and -N═, preferably -NR 1 R 2 and -SH, optionally selected from -NR 3 R 4 , -SH, -≡, -≡N and -N═, or optionally selected from -NR 5 R 6 , -SH, -≡, -≡N and -N═, wherein the fully conjugated compound is directly connected to the hydrogen atom, wherein the substituent R 1 and R 2 has the meaning defined herein, and the substituent R 3 –R 6 are independently selected from -H and alkyl, preferably -H and C1-C4 alkyl.

[0084] Selected from -NR 1 R 2 , -SH, -≡, -≡N and -N═, preferably -NR 1 R 2 The aryl group substituted with one or more substituents of -SH may contain one or more other substituents selected from the group consisting of hydroxy, alkyl, alkoxy, alkylthio, formyl, nitro, halide, hydroxycarbonyl, alkoxycarbonyl and O-containing heteroaryl, and more preferably selected from the group consisting of alkyl, alkoxy, alkylthio, halide and O-containing heteroaryl.

[0085] The N-containing heteroaryl and S-containing heteroaryl may contain one or more other substituents preferably selected from the group consisting of amino, N-alkylamino, N,N-dialkyl-amino, thiol, hydroxy, alkyl, alkoxy, alkylthio, formyl, cyano, isonitrile, ethynyl, nitro, halide, hydroxycarbonyl, alkoxycarbonyl and O-containing heteroaryl, preferably alkyl, alkoxy, alkylthio, halide and O-containing heteroaryl.

[0086] Examples of O-containing heteroaryl groups include, but are not limited to, furyl, benzofuranyl, isobenzofuranyl, oxazolyl, isoxazolyl, benzoxazolyl, and oxadiazolyl.

[0087] Preferred Raman-active reporter compounds are compounds of formula (I),

[0088]

[0089] in

[0090] A 1 、B 1 and C 1 are independently selected from N, CR 16 and CR 17 , the condition is A 1 、B 1 and C 1 Only one of them is N;

[0091] A 2 、B 2 and C 2 are independently selected from N, CR 18 and CR 19 , the condition is A 2 、B 2 and C 2 Only one of them is N;

[0092] E 1 、D 1 、E 2 、D 2 、R 16 、R 17 、R18 and R 19 are independently selected from the group consisting of hydrogen, amino, N-alkylamino, N,N-dialkylamino, thiol, hydroxy, alkyl, alkoxy, alkylthio, formyl, cyano, isonitrile, alkynyl, nitro, halide, hydroxycarbonyl, alkoxycarbonyl and O-containing heteroaryl, preferably selected from the group consisting of hydrogen, alkyl, alkoxy, alkylthio, halide and O-containing heteroaryl; and

[0093] X is a single bond or a linking group selected from the following: 2 -:-CR 8 ═CR 9 -、-N=N-、-≡-、-CR 10 ═CR 11 -o-C6H4-、-CR 10 ═CR 11 -m-C6H4-、-CR 10 ═CR 11 -p-C6H4-、-CR 10 ═CR 11 -o-C6H4-CR 12 ═CR 13 -、-CR 10 ═CR 11 -m-C6H4-CR 12 ═CR 13 -、-CR 10 ═CR 11 -p-C6H4-CR 12 ═CR 13 -、-CR 14 ═NN═CR 15 -,

[0094]

[0095]

[0096] where R 8 –R 15 is selected from hydrogen, alkyl, alkoxy, alkylthio, formyl, cyano, nitro, halide, hydroxycarbonyl and alkoxycarbonyl.

[0097] Preferably, in the general formula (I), the residue A 1 and A 2 is N. More preferably, in the general formula (I), the residue A 1 and A 2 is N, substituent E 1 、D 1 、E 2 、D 2 、R 16 、R17 、R 18 and R 19 For hydrogen.

[0098] Raman-active reporter molecules include, but are not limited to, 2-mercaptopyridine; thiophenol; mercaptobenzoic acid; 4-nitrothiophenol; 3,4-dichlorothiophenol; 3-fluorothiophenol; 4-fluorothiophenol; 3-5-bis(trifluoromethyl)thiophenol; 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)thiophenol, 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-tetrafluorothiophenol, (5-(4-methoxyphenyl)-1,3,4-oxadiazole-2-thiol), (E)-1,2-di(pyridin-4-yl)ethylene, 5-(pyridin-4-yl)-1,3,4-oxadiazole-2-thiol, and 1,4-bis((E)-2-(pyridin-4-yl)vinyl)benzene.

[0099] The first colloid provided in step a) is characterized by a zeta potential value lower than or equal to -25 mV. The second colloid provided in step b) is characterized by a zeta potential value lower than or equal to -25 mV.

[0100] As used herein, the zeta potential value of a colloid refers to the zeta potential value measured on the colloid at a concentration of 0.05 mg nanoparticle material / mL using a Malvern Zetasizer Nano-ZS with a 1 mL folded capillary cell. If necessary, i.e., for colloids having a concentration higher than 0.05 mg nanoparticle material / mL, the colloid was diluted with deionized water to achieve a concentration of 0.05 mg nanoparticle material / mL prior to the zeta potential measurement.

[0101] Preferably, the concentration of nanoparticle material (mg / mL) in the first colloid and the second colloid is less than 0.66 mg / mL, and more preferably between about 0.05 mg / mL and about 0.30 mg / mL, for example, 0.05 mg / mL, 0.10 mg / mL, 0.15 mg / mL, 0.20 mg / mL, 0.25 mg / mL and 0.30 mg / mL.

[0102] After combining the first colloid with the second colloid, the nanoparticles are induced to aggregate, 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 between about 25:1 and about 1:1, preferably about 5:1 and about 1:1, and more preferably about 4:1 and about 3:1. The aggregates described herein are composed 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 aggregation or negligible aggregation of nanoparticles of the second colloid with nanoparticles of the second colloid.

[0103] Induce nanoparticle aggregation by any one of steps d1) to d3) or a combination thereof:

[0104] d1) mixing the third colloid obtained in step c) at a pH between about 2.2 and the lowest pH value at which the net charge of the Raman-active reporter molecule is between 0 and 0.3;

[0105] d2) adding a salt solution, preferably an inorganic salt solution, to the third colloid obtained in step c);

[0106] d3) adding a water-miscible solvent to the third colloid obtained in step c).

[0107] In a preferred embodiment, inducing aggregation of the nanoparticles comprises step d1), i.e., mixing the third colloid obtained in step c) at a pH between about 2.2 and the lowest pH value at which the net charge of the Raman-active reporter molecule is between 0 and 0.3. The net charge of the Raman-active reporter molecule and the pH value corresponding to the net charge can be predicted using online tools such as Chemicalize.com (Chemicalize.ChemAxon.http: / / chemicalize.com / # / calculation). Selective aggregation of the nanoparticles of the first colloid with the nanoparticles of the second colloid occurs at a pH between about 2.2 and the lowest pH value at which the net charge of the Raman-active reporter molecule is between 0 and 0.3.

[0108] Optionally, inducing aggregation of the nanoparticles comprises step d2), i.e., 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 inducing nanoparticle aggregation for those skilled in the art of colloid chemistry (ChemPhysChem 2018, 19, 24-28). Examples of inorganic salts used as a solution (e.g., an aqueous solution) in step d2) of the manufacturing method include, but are not limited to, sodium fluoride, sodium chloride, sodium bromide, sodium iodide, magnesium chloride, potassium chloride, and mixtures thereof.

[0109] In a further alternative embodiment, inducing aggregation of the nanoparticles comprises step d3), i.e., adding a water-miscible solvent to the third colloid obtained in step c). The water-miscible solvent is preferably selected from the group consisting of methanol, ethanol, propanol, isopropanol, tetrahydrofuran, N-methyl-2-pyrrolidone (NMP), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), acetone and acetonitrile.

[0110] The preferred manufacturing method according to the present invention comprises the following steps:

[0111] a) providing a first colloid, the first colloid consisting essentially of nanoparticles having a plasmonic surface and substantially the same size dispersed in an aqueous solvent, and a stabilizer adsorbed on the surfaces of the nanoparticles, and the first colloid having a zeta potential value lower than or equal to -25 mV;

[0112] b) providing a second colloid, the second colloid consisting essentially of nanoparticles having a plasmonic surface and substantially the same size dispersed in an aqueous solvent, a Raman-active reporter molecule of formula (I) adsorbed on the surface of the nanoparticles, and a stabilizer adsorbed on the surface of the nanoparticles, and the second colloid having a zeta potential value less than or equal to -25 mV;

[0113] c) combining the first colloid and 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 between about 25:1 and about 1:1, preferably about 5:1 and about 1:1, more preferably about 4:1 and about 3:1, to provide a third colloid;

[0114] d) inducing aggregation of nanoparticles by any one of steps d1) to d3) or a combination thereof:

[0115] d1) mixing the third colloid obtained in step c) at a pH between about 2.2 and 6.1, preferably between about 2.6 and about 5.7;

[0116] d2) adding a salt solution, preferably an inorganic salt solution, to the third colloid obtained in step c);

[0117] d3) adding a water-miscible solvent to the third colloid obtained in step c); and

[0118] e) Stop gathering.

[0119] Preferably, in the manufacturing method described herein, step b) comprises the following steps performed in the order of b1) to b3):

[0120] b1) providing a colloid comprising nanoparticles having a plasmonic surface and substantially the same size dispersed in an aqueous solvent, and a stabilizer adsorbed on the surface of the nanoparticles, wherein the colloid has a zeta potential value lower than or equal to −25 mV;

[0121] b2) adjusting the pH of the colloid to a value higher than the lowest pH at which the Raman-active reporter molecules adsorbed on the surface of the nanoparticles do not carry a net charge, while maintaining a zeta potential value lower than or equal to -25 mV, preferably lower than -40 mV; and

[0122] b3) adding a solution of a Raman-active reporter molecule in a solvent to the colloid obtained in step b2) while maintaining the zeta potential value lower than or equal to -25 mV.

[0123] The colloid provided in step b1) of the manufacturing method claimed herein consists essentially of nanoparticles having a plasmonic surface and substantially the same size dispersed in an aqueous solvent, and a stabilizer adsorbed on the surface of the nanoparticles. The first colloid provided in step a) of the manufacturing method claimed herein consists essentially of nanoparticles having a plasmonic surface and substantially the same size dispersed in an aqueous solvent, and a stabilizer adsorbed on the surface of the nanoparticles. The second colloid provided in step b) of the manufacturing method claimed herein consists essentially of nanoparticles having a plasmonic surface and substantially the same size dispersed in an aqueous solvent, a Raman-active reporter molecule adsorbed on the surface of the nanoparticles, and a stabilizer adsorbed on the surface of the nanoparticles. Therefore, the nanoparticles of the first colloid and the nanoparticles of the second colloid do not have molecules or organisms present on their surfaces, enabling specific interactions through a lock-and-key mechanism between the nanoparticles of the first colloid and the nanoparticles of the second colloid. Examples of such molecules include antibodies, proteins, antigens, complementary DNA chains, and complementary RNA chains. Examples of such organisms include bacteria, viruses, and spores. Therefore, the first colloid, the second colloid and the colloid provided in step b1) are free of surfactants and polymers and also free of molecules such as antibodies, proteins, antigens, complementary DNA chains and complementary RNA chains and organisms such as bacteria, viruses and spores.

[0124] In step b2) of the manufacturing method according to the present invention, the pH of the colloid is adjusted to a value higher than the lowest pH at which the Raman-active reporter molecules adsorbed on the surface of the nanoparticles do not carry a net charge, while maintaining a zeta potential value below or equal to -25 mV, preferably below -40 mV. The net charge of the Raman-active reporter molecules and the pH value corresponding to the net charge can be predicted using online tools such as Chemicalize.com (Chemicalize.ChemAxon.http: / / chemicalize.com / # / calculation).

[0125] Preferably, in step b2), the pH of the colloid is adjusted to between about 8.2 and about 12.1 while maintaining a zeta potential value lower than or equal to -25 mV, preferably lower than -40 mV. In a more preferred embodiment, in step b2), the pH of the colloid is adjusted to a value of about 11.0.

[0126] In step b3) according to the present invention, a solution of Raman active reporter molecules in a solvent is added to the colloid obtained in step b2) while maintaining a zeta potential value of less than or equal to -25 mV. The solvent for preparing the Raman active reporter molecule solution covers any aqueous solvent and any organic solvent suitable for dissolving the Raman active reporter molecule. Examples of organic solvents include, but are not limited to, alcohols, 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 a mixture of water and a water-miscible solvent, such as methanol, ethanol, propanol, isopropanol, tetrahydrofuran, N- methyl-2-pyrrolidone (NMP), dimethyl sulfoxide (DMSO), N, N- dimethylformamide (DMF), acetone and acetonitrile.

[0127] The preferred manufacturing method according to the present invention comprises the following steps:

[0128] a) providing a first colloid, the first colloid consisting essentially of nanoparticles having a plasmonic surface and substantially the same size dispersed in an aqueous solvent, and a stabilizer adsorbed on the surfaces of the nanoparticles, and the first colloid having a zeta potential value lower than or equal to -25 mV;

[0129] b) providing a second colloid, the second colloid consisting essentially of nanoparticles having a plasmonic surface and substantially the same size dispersed in an aqueous solvent, a Raman-active reporter molecule of formula (I) adsorbed on the surface of the nanoparticles, and a stabilizer adsorbed on the surface of the nanoparticles, and the second colloid having a zeta potential value less than or equal to -25 mV;

[0130] c) combining the first colloid and 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 between about 25:1 and about 1:1, preferably about 5:1 and about 1:1, more preferably about 4:1 and about 3:1, to provide a third colloid;

[0131] d) inducing aggregation of nanoparticles by any one of steps d1) to d3) or a combination thereof:

[0132] d1) mixing the third colloid obtained in step c) at a pH between about 2.2 and 6.1, preferably between about 2.6 and about 5.7;

[0133] d2) adding a salt solution, preferably an inorganic salt solution, to the third colloid obtained in step c);

[0134] d3) adding a water-miscible solvent to the third colloid obtained in step c); and

[0135] e) stop gathering,

[0136] Wherein step b) comprises the following steps performed in the order of b1) to b3):

[0137] b1) providing a colloid consisting essentially of nanoparticles having a plasmonic surface and substantially the same size dispersed in an aqueous solvent, and a stabilizer adsorbed on the surface of the nanoparticles, wherein the colloid has a zeta potential value lower than or equal to −25 mV;

[0138] b2) adjusting the pH of the colloid to between about 8.0 and about 12.1, preferably between about 8.2 and about 12.1, while maintaining a zeta potential value below or equal to -25 mV, preferably below -40 mV; and

[0139] b3) adding a solution of a Raman-active reporter molecule of formula (I) in a solvent to the colloid obtained in step b2) while maintaining the zeta potential value lower than or equal to -25 mV.

[0140] The inventors have found that the signal intensity of the SERS tag manufactured according to the present invention can be further improved by ensuring that a sub-monolayer or monolayer of Raman active reporter molecules is adsorbed on the surface of the nanoparticles contained in the second colloid. Therefore, a further preferred embodiment according to the present invention relates to a method for manufacturing a SERS tag as claimed in the present invention, wherein the nanoparticles of the second colloid have adsorbed on their surface a sub-monolayer or monolayer of Raman active reporter molecules. As used herein, a monolayer of Raman active reporter molecules adsorbed on the surface of the nanoparticles of the second colloid refers to a layer of the thickness of one Raman active reporter molecule adsorbed on the surface of the nanoparticles. As used herein, a sub-monolayer of Raman active reporter molecules refers to an incomplete monolayer of Raman active reporter molecules. In order to ensure that a sub-monolayer or monolayer of Raman active reporter molecules is adsorbed on the surface of the nanoparticles of the second colloid, in step b3), the amount of Raman active reporter molecules added to the colloid obtained in step b2) must be calculated according to the shape and size of the nanoparticles of the second colloid by methods well known to those skilled in the art.

[0141] The manufacturing method claimed herein can also prepare SERS tags comprising a mixture of different Raman-active reporter molecules (i.e., a mixture of two or more different Raman-active reporter molecules). This is particularly advantageous because it enables a variety of SERS tags to be obtained by combining a limited number of different Raman-active reporter molecules in different proportions, wherein each of the SERS tags is characterized by a unique SERS signal. In order to achieve such a SERS tag, nanoparticles of the second colloid provided in step b) are prepared so that a mixture of different Raman-active reporter molecules (i.e., a mixture of two or more different Raman-active reporter molecules) is adsorbed on their surface. Such a second colloid can be prepared by using a solution of Raman-active molecules in a solvent comprising two or more different Raman-active reporter molecules in step b3) of the manufacturing method, or by continuously performing step b3) of the manufacturing method and using different solutions comprising different Raman-active reporter molecules each time. Therefore, a further embodiment according to the present invention relates to a manufacturing method, wherein in step b3), the solution of Raman-active reporter molecules in a solvent comprises a mixture of two or more different Raman-active reporter molecules. According to another further embodiment of the present invention, a method of manufacturing is provided wherein step b3) is performed n times in succession, n ≥ 2, each time using a solution comprising a Raman-active reporter molecule that is structurally different from the Raman-active reporter molecule used in the remaining n-1 solutions used in the remaining n-1 steps. As used herein, different Raman-active reporter molecules refer to Raman-active reporter molecules that have different chemical structures and provide different SERS spectra.

[0142] In order to further increase the number of low number aggregates, and thereby increase the intensity of the SERS signal provided by the SERS tag manufactured by the manufacturing method claimed herein, it is preferred 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 between about 5:1 and about 1:1, preferably about 4:1 and about 3:1. Figure 3b It was demonstrated that the ratio of the number of nanoparticles of the first colloid to the number of nanoparticles of the second colloid was between about 5:1 and about 1:1, which allowed obtaining SERS tags with a large number of low-number aggregates (such as dimers, trimers, and tetramers).

[0143] In order to induce aggregation by the method described in step d1), the third colloid obtained in step c) needs to be mixed at a pH between about 2.2 and the lowest pH value at which the net charge of the Raman-active reporter molecule is between 0 and 0.3. For the Raman-active reporter molecule of general formula (I), the lowest pH value is a pH between about 2.2 and 6.1, preferably between about 2.6 and about 5.7. This can be achieved by adding an acid solution to the third colloid obtained in step c), or by adjusting the pH of the first colloid so that the colloid obtained in step d1) has the desired pH value.

[0144] Therefore, an embodiment according to the present invention relates to a manufacturing method, wherein step d1) further comprises adding an acid solution to the third colloid obtained in step c) while mixing, so that the pH of the resulting colloid is between about 2.2 and the lowest pH value at which the net charge of the Raman-active reporter molecule is between 0 and 0.3, which in the case of the Raman-active reporter molecule of formula (I) is a pH between about 2.2 and 6.1, preferably between about 2.6 and about 5.7. Suitable acid solutions include, but are not limited to, acetic acid, hydrochloric acid, and nitric acid.

[0145] In order to avoid additional manufacturing steps, it is convenient to adjust the pH of the first colloid so that the colloid obtained in step d1) has a pH between about 2.2 and the lowest pH value at which the net charge of the Raman-active reporter molecule is between 0 and 0.3, which in the case of the Raman-active reporter molecule of general formula (I) is a pH between about 2.2 and 6.1, preferably between about 2.6 and about 5.7.

[0146] The manufacturing method claimed herein further comprises step e) stopping the aggregation. Preferably, step e) comprises any one of the following steps e1)-e4):

[0147] e1) adjusting the pH of the colloid obtained in step d) to a value higher than the lowest pH value at which the Raman-active reporter molecules adsorbed on the surface of the nanoparticles do not carry a net charge;

[0148] e2) diluting the colloid obtained in step d) with water, preferably such that the concentration of nanoparticles in the colloid is less than 6*10 9 nanoparticles / mL;

[0149] e3) adding a polymer to the colloid obtained in step d);

[0150] e4) adding a dielectric material precursor to the colloid obtained in step d).

[0151] In the manufacturing method of the invention claimed herein, aggregation can be stopped by any one of methods e1) to e4).

[0152] As described in step e1), the pH of the colloid obtained in step d) is adjusted to a value higher than the lowest pH at which the Raman-active reporter molecules adsorbed on the surface of the nanoparticles do not carry a net charge, thereby increasing the electrostatic repulsion between the nanoparticle aggregates and thus halting the aggregation process. The net charge of the Raman-active reporter molecules and the pH corresponding to the net charge can be predicted using online tools such as Chemicalize.com (Chemicalize.ChemAxon. http: / / chemicalize.com / # / calculation).

[0153] As described in step e3), adding a polymer to the colloid obtained in step d) increases the steric repulsion between the nanoparticle aggregates and stops the aggregation process. Advantageously, the polymer used in step e3) does not affect the SERS signal presented by the SERS tag. Suitable polymers include, but are not limited to, polyvinyl pyrrolidone and polyethylene glycol.

[0154] As described in step e4), adding a dielectric material precursor to the colloid obtained in step d) results in encapsulating the nanoparticle aggregates with at least one layer of dielectric material, which stabilizes the SERS tag and implicitly causes the aggregation process to stop. Preferably, the dielectric material precursor is a silica precursor. Silica precursors include, but are not limited to, an ethanolic solution of tetraethyl orthosilicate and 3-aminopropyltrimethoxysilane, an ethanolic solution of tetraethyl orthosilicate and (3-mercaptopropyl)trimethoxysilane, an aqueous solution of sodium silicate, and an aqueous solution of (3-mercaptopropyl)trimethoxysilane.

[0155] Optionally, the aggregation process can be stopped by diluting the colloid obtained in step d) with water. Preferably, the aggregation is stopped by diluting the colloid obtained in step d) with water, so as to reach a concentration of nanoparticle material [μg / mL] lower than or equal to 12.5 μg / mL (see, for example: E1-E8, E11-E14). Optionally, the aggregation is stopped by diluting the colloid obtained in step d) with water, so as to reach a nanoparticle concentration lower than 6*10 9 nanoparticles / mL.

[0156] A further preferred embodiment of the present invention relates to a method for producing a SERS tag, wherein step e) consists essentially of step e3), and the method further comprises diluting the colloid obtained in step e3) with water and / or coating the SERS tag with a dielectric material.

[0157] In a further preferred embodiment, steps c) and d) of the manufacturing method claimed herein are carried out simultaneously in a continuous flow system. In this particular manufacturing method, a continuous flow reactor is used. This continuous flow reactor is Figure 1b and Figure 1c Schematically represented in, and includes two pressurized tanks (111b, 112b, 111c, 112c) connected to a three-way joint (113b, 113c) and a collection container (114b, 114c) via a pipeline. The second colloid is stored in the pressurized tanks (111b, 111c), and the first colloid is stored in the pressurized tanks (112b, 112c). A container (114b, 114c) open to the atmosphere is used to collect the SERS tags and optionally stop aggregation (114c). The pipeline of each pressurized tank (111b, 112b, 111c, 112c) connected to the three-way joint (113b, 113c) is used for colloid transport. Another pipeline of the three-way joint (113b, 113c) connected to the collection container (114b, 114c) is used for aggregation. Nanoparticle aggregates obtained by aggregation are collected in a collection container (114b), where the aggregation is stopped.

[0158] When steps c) and d) of the manufacturing method are simultaneously performed in a continuous flow system, it is further preferred that step e) is also performed in the continuous flow system.

[0159] As described above, the nanoparticles used in the method for manufacturing a SERS tag can have any shape that can be manufactured, such as a sphere, a spheroid, a rod, a disk, a prism, and a cube. Preferably, the shape of the nanoparticles having a plasmonic surface used in the manufacturing method of the present invention claimed herein is selected from the group consisting of a sphere and a spheroid. Even more preferably, the nanoparticles have a spheroidal shape.

[0160] In a preferred embodiment, the nanoparticles of the first colloid and the nanoparticles of the second colloid have the same size. Conveniently, for this method of manufacturing a SERS tag, the colloid used to prepare the second colloid (ie, the colloid provided in step b1)) is the first colloid.

[0161] 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 of the first colloid can be smaller than the size of the nanoparticles of the second colloid, thereby resulting in a SERS tag having the same Figure 3a Alternatively, the size of the nanoparticles in the first colloid can be larger than that of the nanoparticles in the second colloid, resulting in a SERS tag with a similar structure to that depicted. Figure 3b The depicted SERS tag has a similar structure.

[0162] The manufacturing method claimed herein is preferably carried out using colloids, wherein the plasmonic surface of the nanoparticles in the first colloid and / or the plasmonic surface of the nanoparticles in the second colloid are made of gold, more preferably using colloids comprising solid gold nanoparticles, and even more preferably using citrate-stabilized gold colloids.

[0163] Example

[0164] The present invention will now be described in more detail with reference to the following non-limiting examples.

[0165] generally

[0166] The following reagents were provided by the following suppliers:

[0167] Gold chloroaurate trihydrate (>99.9%; CAS No.: 16961-25-4), sodium borohydride (99.99%; CAS No.: 16940-66-2), trisodium citrate dihydrate (≥99.5%; CAS No.: 6132-04-3), (E)-1,2-di(pyridin-4-yl)ethylene (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 99%; CAS No.: 623-27-8), 4-methylpyridine (99%; CAS No.: 108-89-4), acetic anhydride (Reagent Plus, ≥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 grade, 34-37%; CAS No.: 7647-01-0) was purchased from Fisher Scientific.

[0168] Zeta potential values ​​were measured using a Malvern Zetasizer Nano-ZS (DTS1060) with a 1 mL collapsed capillary chamber. Optical absorption spectra were recorded on an Agilent 8453 spectrophotometer and a Perkin Elmer Lambda 650.

[0169] Scanning electron microscope (SEM) images were taken on a Hitachi S-4500.

[0170] Raman spectra were acquired on an Ocean Optics QE 6500 at 785 nm.

[0171] Nominal 140 nm gold colloidal particles were sized by sending samples to EAG Laboratories for transmission electron microscopy (TEM) imaging. Images were analyzed using ImageJ software (https: / / imagej.nih.gov / ij / ). A batch was considered nominally 140 nm if the number average particle size was within ±9 nm of the nominal diameter, where the number average particle size was determined by measuring 230 individual particles in TEM micrographs. The size-related characteristic selected to describe individual particles was the "circular equivalent" (CE) diameter, which corresponds to the diameter of a circle having the same area as the orthographic projection of the particle.

[0172] The gold nanoparticle diameter d (nm) of the 40, 60, and 90 nm nanoparticle batches was calculated by the method of Haiss and colleagues (Anal. Chem. 2007, 79, 4215-4221) using the following formula:

[0173]

[0174] where λ spr is the surface plasmon resonance peak position in the extinction pattern of the colloidal sample on a Perkin Elmer Lambda 650 UV Vis. Batches are considered nominally 40 nm, 60 nm, or 90 nm if the surface plasmon resonance peak position correlates to a diameter within ±9 nm of the nominal diameter.

[0175] I. Preparation of Au colloid stock solutions (S1-S4).

[0176] Gold colloid stock solutions (S1-S4) characterized by the Au nanoparticle size (nm), Au concentration (mg / mL), and pH values ​​shown in Table 1 were prepared as follows:

[0177] I.1 Preparation of 40 nm gold colloid stock solution (S1)

[0178] In a completely clean 100 L glass reactor (ChemGlass) with a jacket, 79.5 L of 17 MΩ water were cooled to 3.5 ± 0.5° C. While stirring with an impeller at 400 rpm, a 20 wt % aqueous solution of gold chloroaurate trihydrate (100 g) was added.

[0179] A 30 wt % aqueous solution of trisodium citrate dihydrate (174.3 g) and a 23.5 wt % aqueous solution of hydroxylamine hydrochloride (155.6 g) were combined and added to the reactor. After 10 seconds, 800 μL of sodium borohydride as a 0.063 wt % 0.01 N sodium hydroxide solution was injected into the reactor. The reagents were allowed to react for 2 minutes and then drained into a clean drum to provide a 40 nm Au colloidal stock solution (S1) with a gold concentration of 0.25 mg Au / mL and a pH of approximately 2.4.

[0180] I.2 Preparation of 60 nm gold colloid stock solution (S2)

[0181] In a completely clean 100 L glass reactor (ChemGlass) with a jacket, 79.5 L of 17 MΩ water were cooled to 3.5 ± 0.5° C. While stirring with an impeller at 400 rpm, a 20 wt % aqueous solution of gold chloroaurate trihydrate (200 g) was added.

[0182] A 30 wt% aqueous solution of trisodium citrate dihydrate (173.3 g) and a 17.5 wt% aqueous solution of hydroxylamine hydrochloride (217.1 g) were combined and added to the reactor. After 10 seconds, 800 μL of sodium borohydride as a 0.052 wt% 0.01 N sodium hydroxide solution was injected into the reactor. The reagents were allowed to react for 2 minutes and then drained into a clean drum. The batch was diluted to 160 L with 17 MΩ water to obtain a 60 nm Au colloidal stock solution (S2) with a gold concentration of 0.25 mg Au / mL and a pH of approximately 2.4.

[0183] I.3 Preparation of 90 nm gold colloid stock solution (S3)

[0184] In a completely clean 100 L glass reactor (ChemGlass) with a jacket, 79.5 L of 17 MΩ water was cooled to 3.5 ± 0.5° C. While stirring with an impeller at 400 rpm, a 20 wt % aqueous solution of gold chloroaurate trihydrate (200 g) was added.

[0185] A 30 wt% aqueous solution of trisodium citrate dihydrate (173.3 g) and a 17.5 wt% aqueous solution of hydroxylamine hydrochloride (217.1 g) were combined and added to the reactor. After 10 seconds, 900 μL of sodium borohydride as a 0.01 wt% 0.01 N sodium hydroxide solution was injected into the reactor. The reagents were allowed to react for 2 minutes and then drained into a clean drum. The batch was diluted to 160 L with 17 MΩ water to obtain a 90 nm Au colloidal stock solution (S3) with a gold concentration of 0.25 mg Au / mL and a pH of approximately 2.4.

[0186] I.4 Preparation of 140 nm gold colloid stock solution (S4)

[0187] In a 2 L glass jar, 1.5 L of 17 MΩ water was stirred at room temperature, and a 20 wt% aqueous solution of gold chloroaurate trihydrate (2.5 g) was added while stirring.

[0188] A 30.7 wt% aqueous solution of trisodium citrate dihydrate (15.37 g) and a 16.7 wt% aqueous solution of hydroxylamine hydrochloride (8.37 g) were combined and added to the reactor. Five seconds later, 25 μL of sodium borohydride as a 0.01 wt% 0.01 N sodium hydroxide solution was injected into the reactor. Stirring for an additional 15 minutes yielded a 140 nm Au colloidal stock solution (S4) with a gold concentration of 0.25 mg Au / mL and a pH of approximately 2.4.

[0189] Table 1: Characterization of Au colloid stock solutions (S1–S4).

[0190]

[0191] II. Preparation of the first colloid (A1-A9) (step a) of the manufacturing method)

[0192] Starting from Au colloid solutions S1, S2, and S4, the first colloids (A1–A9) were obtained. If necessary (e.g., Au colloids A1 and A7), the Au colloid stock solutions were diluted with deionized water to obtain the Au concentrations (mg / mL) shown in Table 2. Furthermore, if necessary (e.g., Au colloids A3, A4, A5, A6, A8, A9), the Au colloid stock solutions, either as is or after dilution, were treated with 0.1 mM aqueous NaOH or 0.1 mM aqueous HCl to adjust the pH of the first colloids to the pH values ​​shown in Table 2.

[0193] Table 2: Characterization of the first colloids (A1–A9).

[0194]

[0195] Only stable first colloids (i.e., colloids that have not aggregated) are suitable for use in the manufacturing method according to the present invention. First, colloids A1-A9 are stable, i.e., no aggregation of gold nanoparticles is detected by visual inspection of the color change from pink to purple. Further, as Figure 4 It was confirmed that various Au colloids characterized by having a zeta potential value lower than or equal to -25 mV as measured as described in the following Item IV are stable. Therefore, such Au colloids can also be used as the first colloid in the manufacturing method according to the present invention.

[0196] III. A second method comprising Au nanoparticles and Raman-active reporter molecules adsorbed on the surface of the Au nanoparticles Preparation of colloids (D1–D11) (step b) of the manufacturing method)

[0197] Starting from Au colloid stock solutions S1–S3, Au colloids (B1–B8) characterized by Au concentration (mg / mL) and pH values ​​as shown in Table 3 were prepared. If necessary (e.g., Au colloids B1 and B5), the preparation involved diluting the Au colloid stock solution with deionized water to obtain the indicated Au concentration (mg / mL) and adjusting the pH by adding 1 M NaOH aqueous solution to the Au colloid stock solution itself or after dilution.

[0198] Table 3: Characterization of Au colloids B1–B9.

[0199]

[0200] The Raman-active reporter molecule 1,4-bis((E)-2-(pyridin-4-yl)vinyl)benzene was synthesized as follows:

[0201] Terephthalaldehyde (3.44 g, 25.7 mmol), 4-picoline (9.57 g, 103 mmol) and acetic anhydride (25 mL) were added to a 50 ml round bottom flask with a stirring bar. The mixture was refluxed until the aldehyde was no longer present (4 hours, checked by TLC). The reaction was cooled to room temperature and quenched by pouring it into 100 ml of ice water. The cold mixture was neutralized to pH 7 using 6N aqueous NaOH solution, and the resulting brown precipitate was filtered, washed with water and air dried. It was extracted with dichloromethane and the solvent was then concentrated to dryness to give the crude product, which was purified by flash column chromatography on silica gel (methanol / dichloromethane: 5 / 95) to give 0.875 g of the target Raman active reporter molecule (12%) as a yellow solid.

[0202] Second colloids (D1–D11) containing Au nanoparticles with Raman-active reporter molecules adsorbed on their surfaces were prepared by adding a 0.1 mM volume of an ethanolic solution of the Raman-active reporter molecules to 20 mL of Au colloids (B1–B5, B9) or 3 L of Au colloid (B8), followed by stirring the resulting mixture at room temperature for 30 minutes (second colloids D1–D7, D9–D11) or 1 hour (second colloid D8). Table 4 provides a summary of the volumes of the Raman-active reporter molecules and ethanolic solutions of the Raman-active reporter molecules used in the preparation of second colloids D1–D11.

[0203] Table 4: Preparation of the second colloids D1–D11.

[0204]

[0205] Treatment of 20 ml of Au colloids B6 and B7 at a pH below the lowest pH at which (E)-1,2-di(pyridin-4-yl)ethylene carries no net charge, as predicted by Chemicalize.com, with 750 μL of a 0.1 mM solution of (E)-1,2-di(pyridin-4-yl)ethylene in ethanol, followed by stirring of the resulting mixture at room temperature for 30 minutes, resulted in unstable colloids, as indicated by a visual inspection of the color change of the mixture from pink to purple during stirring. Unstable colloids cannot be used in the manufacturing method according to the present invention. To ensure the stability of the second colloid, i.e., to avoid aggregation of the Au nanoparticles contained in the colloid, it is important that during the preparation and storage of the colloid, the zeta potential value is less than or equal to −25 mV and the pH value is above the lowest pH at which the Raman-active reporter molecules adsorbed on the Au nanoparticles carry no net charge, as predicted, for example, by Chemicalize.com. In this sense, prior to treatment with the Raman-active reporter molecules, the pH of the Au colloids (B1-B5, B8, B9) used to prepare the second colloid is adjusted to a value higher than the lowest pH value at which the Raman-active reporter molecules adsorbed on the Au nanoparticles contained in the Au colloids (B1-B5, B8, B9) do not carry a net charge, wherein said value can be predicted, for example, using Chemicalize.com.

[0206] IV. Stability of the first and second colloids. In order to be applicable to the manufacturing method of providing a SERS tag having a large number of small-sized aggregates according to the present invention, the first and second colloids must be stable, i.e., nanoparticle aggregation of the nanoparticles must be avoided during the preparation and storage of the colloids. This can be ensured by maintaining the zeta potential value of the colloid at a value lower than or equal to -25 mV. Figure 4 As shown in Table 5, in the method for manufacturing a SERS tag according to the present invention, various Au colloids can be used as the first and second colloids.

[0207] The zeta 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 reported in Table 5 and plotted in Table 5. Figure 4 middle.

[0208] Zeta potential measurements were performed on a Malvern Zetasizer Nano-ZS (DTS1060) with a 1 mL collapsed capillary chamber. Water and gold at 25°C were pre-loaded onto the instrument, and 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.

[0209] The zeta potential values ​​of the first colloid A2 and the second colloids D2, D5, and D6 at approximately the pH used for the synthesis of SERS tags according to the present invention are reported in column 2 of Table 5. The zeta potential values ​​of the first colloid A2 and the second colloids D2, D5, and D6 at the pH at which the colloids became unstable are reported in column 3 of Table 5. The instability of the colloids was determined by visually inspecting the color change of the colloids from pink to purple.

[0210] Table 5: Zeta potential measurements of the first and second colloids.

[0211]

[0212] V. Combining the first colloid and the second colloid, inducing aggregation and stopping aggregation (steps c), d) and e) of the manufacturing method)

[0213] V.1 SERS tags containing Au nanoparticles of the same size (Examples E1–E8, E12–E14)

[0214] V.1.a Batch Aggregation (Examples E1-E7, E12-E14)

[0215] The second colloid (D1–D6, D9–D11, 20 mL) was quickly poured into 80 mL of the first colloid (A1–A5, A9), and the mixture was stirred in a mixing vessel with a magnetic stir bar. 30 seconds after the colloids were combined, a 125 μL aliquot was sampled and diluted to 1 mL with water to stop aggregation. At this dilution (12.5 μg Au / mL, corresponding to approximately 5.72*10 9 The aggregation was stopped at 100 nm Au nanoparticles / mL, and the SERS signal was measured using a QE65000 spectrometer purchased from OceanOptics Inc. with 785 nm laser excitation and a one-second integration time. The results of the SERS signal measurement of the SERS tags fabricated as described above are shown in Table 6.

[0216] As demonstrated by Examples E1-E7 and E12-E14 and Comparative Examples C1-C3 according to the inventive method claimed herein, selective aggregation of particles of the first colloid and particles of the second colloid does not occur at low pH values ​​(e.g., 1.8) and at pH values ​​higher than the lowest pH value where the net charge of the Raman-active reporter molecule is between 0 and 0.3 as predicted by Chemicalize.com.

[0217] Table 6: SERS signal intensities displayed by the SERS tags according to Examples E1-E7, E12 and Comparative Examples C1-C3.

[0218]

[0219] V.1.b Continuous Flow System Aggregation (Example E8)

[0220] A schematic diagram of the continuous flow reactor used in the production method according to the present invention is shown in Figure 1b The pressurized tank (111b) holds the second colloid, and the second pressurized tank (112b) holds the first colloid. A container (114b) open to the atmosphere is used to collect the SERS tags and contains a concentrated polymer solution for stopping the aggregation reaction. Clear fluorinated ethylene propylene (FEP) tubing (8 mm nominal ID) from Scientific Experts was used for colloid transport. A 30 cm length of FEP tubing was used to connect the pressurized tank (111b) to the tee (113b). A 30 cm length of FEP tubing was used to connect the pressurized tank (112b) to the tee (113b). A 7 m length of FEP tubing was used to connect the tee (113b) to the collection container (114b). The section of tubing between the tee (113b) and the collection container (114b) is where the colloid will accumulate.

[0221] The above colloids A8 (12 L) and D8 (3 L) were placed in tanks 112b and 111b respectively. The tank (111b) containing the second colloid (D8) was pressurized so that the colloid flow rate was 0.66 L / min. The tank (112b) containing the first colloid (A8) was pressurized so that the colloid flow rate was 2.65 L / min. After combining at the three-way joint, the pH value of the combined colloids (characterized by the ratio of the number of nanoparticles of the first colloid A8 to the number of nanoparticles of the second colloid D8 being ≈4:1) was about 4.8 and the flow rate in the focusing tube was 3.31 L / min, resulting in a linear velocity of 1 m / s. SERS measurements were performed through a transparent FEP tube at the 1 m mark along the length of the focusing tube, and the probe was coupled to an optical cable to an Ocean Optics QE65000 spectrometer with 785 nm excitation. The integration time was shortened so that the measurement at the end mark of the tube was equal to the value obtained from a 50 μL aliquot sampled at the opening of the tube and diluted with water to 1 mL (12.5 μg Au / mL), i.e., where the sample was dilute with water to stop aggregation and measured using an Ocean Optics QE65000 spectrometer set to a one-second integration time and 785 nm laser excitation. The results of the SERS measurements are given in Figure 5 The SERS signal intensity of the sample collected at the end of the tube and measured at 12.5 μg Au / mL was 11044 counts per second [±500].

[0222] V.2 First colloid and second colloid containing Au nanoparticles of different sizes (Examples E9-E11)

[0223] V.2.a Batch Aggregation (Examples E9 and E10)

[0224] Example E9

[0225] The second colloid (D7, 30 mL) was quickly poured into the first colloid (A1, 55 mL) and the mixture was stirred in a mixing container with a magnetic stirring bar. 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 mixing for 30 seconds at a pH of about 2.9, the aggregation was stopped by adding a polymer solution, and the resulting aggregates were further coated with silica to provide the target SERS tag as described in US8497131B2. A ~2 μL aliquot of the SERS tag was dropped onto a silica wafer and dried. The sample was imaged on a Hitachi S-4500 field emission SEM and is shown in FIG. Figure 3a The SERS signal was measured using a QE65000 spectrometer purchased from OceanOptics Inc. with 785 nm laser excitation and a one-second integration time. The SERS signal intensity measured at 12.5 μg Au / mL was 18,377 counts per second [±500].

[0226] Example E10

[0227] The second colloid (D7, 20 mL) was quickly poured into the first colloid (A7, 200 mL), and the mixture was stirred in a mixing container with a magnetic stirring bar. The ratio of the number of nanoparticles of the first colloid to the number of nanoparticles of the second colloid was ≈2.65:1.

[0228] After mixing for 30 seconds at a pH of approximately 2.9, aggregation was stopped by adding the polymer solution and the resulting aggregates were coated with silica to provide the target SERS tags as described in US Pat. No. 8,497,131 B2. A ~2 μL aliquot of the SERS tags was drop-cast onto a silica wafer and dried. The samples were imaged on a Hitachi S-4500 field emission SEM and are shown in FIG. Figure 3b .

[0229] V.2.b Flow system aggregation (Example E11)

[0230] A schematic diagram of the continuous flow reactor used in the production method according to the present invention is shown in Figure 1b The pressurized tank (111b) holds the second colloid, and the second pressurized tank (112b) holds the first colloid. A container open to the atmosphere (collection container, 114b) is used to collect the SERS tags and stop the aggregation. Fluorinated ethylene propylene (FEP) tubing (8 mm nominal ID) from Scientific Experts was used for colloid transport. A 30 cm piece of clear FEP tubing was used to connect the pressurized tank (111b) to the tee (113b). A 30 cm piece of FEP tubing was used to connect the pressurized tank (112b) to the tee (113b). A 7 m piece of FEP tubing was used to connect the tee (113b) to the collection container (114b). The section of tubing between the tee (113b) and the collection container (114b) is where the colloid will accumulate.

[0231] The above colloids A6 (12 L) and D8 (3 L) were placed in tanks 112b and 111b respectively. The tank (111b) containing the second colloid (D8) was pressurized so that the colloid flow rate was 0.66 L / min. The tank (112b) containing the first colloid (A6) was pressurized so that the activated colloid flow rate was 2.65 L / min. After combining at the three-way joint, the pH of the combined colloids (characterized by the ratio of the number of nanoparticles of the first colloid A6 to the number of nanoparticles of the second colloid D8 being ≈1.2:1) was about 4.8 and the flow rate in the focusing tube was 3.31 L / min, resulting in a linear velocity of 1 m / s. SERS measurements were performed through a transparent FEP tube at the 1 m mark along the length of the focusing tube, with the probe coupled to an optical cable to an Ocean Optics QE65000 spectrometer with 785 nm excitation. The integration time was shortened so that the measurement at the end mark of the tube was equal to the value obtained from a 50 μL aliquot sampled at the opening of the tube and diluted to 1 mL (12.5 μg Au / mL) with water (step e2) and measured using an Ocean Optics QE65000 spectrometer set to a one second integration time using 785 nm laser excitation. The results of these measurements are given in Figure 5 The SERS signal intensity of the sample collected at the end of the tube and measured at 12.5 μg Au / mL was 28303 counts per second [±500].

Claims

1. A method for manufacturing a surface-enhanced Raman spectroscopy (SERS) tag, comprising the following steps: a) providing a first colloid, the first colloid consisting essentially of nanoparticles having a plasmonic surface and substantially the same size dispersed in an aqueous solvent, and a stabilizer adsorbed on the surfaces of the nanoparticles, and the first colloid having a zeta potential value lower than or equal to -25 mV; b) providing a second colloid consisting essentially of nanoparticles having a plasmonic surface and substantially the same size dispersed in an aqueous solvent, a Raman-active reporter molecule adsorbed on the surface of the nanoparticles, and a stabilizer adsorbed on the surface of the nanoparticles, and having a zeta potential value less than or equal to -25 mV; c) combining the first colloid and the second colloid so that the ratio of the number of nanoparticles of the first colloid to the number of nanoparticles of the second colloid is between 25:1 and 1:1 to provide a third colloid; d) inducing aggregation of nanoparticles by any one of steps d1) to d3) or a combination thereof: d1) mixing the third colloid obtained in step c) at a pH between 2.2 and the lowest pH value at which the net charge of the Raman-active reporter molecule is between 0 and 0.3; d2) adding a saline solution to the third colloid obtained in step c); d3) adding a water-miscible solvent to the third colloid obtained in step c); and e) Stop gathering.

2. The method according to claim 1, wherein step b) comprises the following steps performed in the order of b1) to b3): b1) providing a colloid consisting essentially of nanoparticles having a plasmonic surface and substantially the same size dispersed in an aqueous solvent, and a stabilizer adsorbed on the surface of the nanoparticles, wherein the colloid has a zeta potential value lower than or equal to −25 mV; b2) adjusting the pH of the colloid to a value higher than the lowest pH value at which the Raman-active reporter molecules adsorbed on the surface of the nanoparticles do not carry a net charge, while maintaining the zeta potential value lower than or equal to -25 mV; and b3) adding a solution of the Raman-active reporter molecule in a solvent to the colloid obtained in step b2) while maintaining the zeta potential value lower than or equal to -25 mV.

3. The method of claim 1 or 2, wherein the stabilizer is selected from the group consisting of carboxylic acids, carboxylates, phosphoric acid, phosphates, ascorbic acid, ascorbates, and mixtures thereof.

4. The method according to claim 1 or 2, wherein the nanoparticles of the second colloid have adsorbed on their surface a sub-monolayer or monolayer of Raman-active reporter molecules. 5 . The method according to claim 2 , wherein in step b3), the solution of the Raman-active reporter molecule in the solvent comprises a mixture of two or more different Raman-active reporter molecules. 6 . The method according to claim 1 , wherein in step c), the ratio of the number of nanoparticles of the first colloid to the number of nanoparticles of the second colloid is between 4:1 and 3:

1.

7. The method according to claim 1 or 2, wherein step d1) further comprises adding an acid solution to the third colloid obtained in step c) while mixing, so that the pH value of the resulting colloid is between 2.2 and the lowest pH value at which the net charge of the Raman-active reporter molecule is between 0 and 0.

3.

8. The method according to claim 1 or 2, wherein the pH of the first colloid is adjusted so that the pH of the third colloid obtained in step d1) is between 2.2 and the lowest pH value at which the net charge of the Raman-active reporter molecule is between 0 and 0.

3.

9. The method according to claim 1 or 2, wherein step e) comprises any one of the following steps e1) to e4): e1) adjusting the pH of the colloid obtained in step d) to a value higher than the lowest pH value at which the Raman-active reporter molecules adsorbed on the surface of the nanoparticles do not carry a net charge; e2) diluting the colloid obtained in step d) with water; e3) adding a polymer to the colloid obtained in step d); e4) adding a dielectric material precursor to the colloid obtained in step d).

10. The method according to claim 9, wherein step e) consists essentially of step e3), and the manufacturing method further comprises diluting the colloid obtained in step e3) with water and / or coating the SERS tag with a dielectric material.

11. The method according to claim 1 or 2, wherein steps c) and d) are performed simultaneously in a continuous flow system.

12. The method of claim 11, wherein step e) is performed in a continuous flow system.

13. The method according to claim 1 or 2, wherein the nanoparticles of the first colloid and the nanoparticles of the second colloid have the same size.

14. The method of claim 1 or 2, wherein the nanoparticle size of the first colloid is different from the nanoparticle size of the second colloid. 15 . The method according to claim 1 , wherein the plasmonic surface of the nanoparticles in the first colloid and / or the plasmonic surface of the nanoparticles in the second colloid are made of gold.

Images