OA20528A

OA20528A - Method for manufacturing surface enhanced raman spectroscopy tags

Info

Publication number: OA20528A
Application number: OA1202200012
Authority: OA
Inventors: Marcelo Eduardo Piotti; Raymond H SCHEFFLER; Mark Kowalski
Original assignee: Sicpa Holding Sa
Priority date: 2019-07-15
Filing date: 2020-07-13
Publication date: 2022-09-30

Abstract

The present invention relates to the field of methods of manufacturing of surface enhanced Raman spectroscopy (SERS) tags. The manufacturing method according to the present invention is reproducible and versatile and enables the production in an expedient manner of high quantities of SERS tags characterized by a narrow size distribution and a high ratio of lownumber aggregates. SERS tags manufactured by the inventive manufacturing method described herein provide increased ensemble SERS responses

Description

Method for Manufacturing Surface Enhanced Raman Spectroscopy Tags

FIELD OF THE INVENTION

The présent invention relates to the field of methods of manufacturing of surface enhanced Raman spectroscopy (SERS) tags. The manufacturing method according to the présent invention is reproducible and versatile and enables the production in an expédient manner of high quantities of SERS tags characterized by a narrow size distribution and a high ratio of low-number aggregates. SERS tags manufactured by the inventive manufacturing method described herein provide increased ensemble SERS responses.

BACKGROUND OF THE INVENTION

Surface enhanced Raman spectroscopy (SERS) tags hâve proved useful for a variety of applications including product labeliing for identification and authentî cation purposes, and hîghthroughput multiplex screening in microarray technology, diagnosis and bioimaging. SERS tags are aggregates of nanoparticles presenting a plasmonic surface and having Raman active reporter molécules adsorbed on their surface. The nanoparticles presenting a plasmonic surface are responsible for the génération of the electric field required for the Raman amplification, while the Raman active reporter molécule provides the unique vibrational fingerprint of the SERS tag. Typically, the aggregates présent an extenial coating layer which a) isolâtes the SERS tag from the extenial medium thereby, preventing the Raman active reporter molécules from leaching out from the SERS tag and protecting the SERS tag from contaminations of the extenial medium that may give rise to vibrational noise, b) increases the colloïdal stability of the SERS tag, and c) provides a convenient surface for further Chemical functionalization. To date, polymers and silica hâve been employed as extenial coating layers.

Duc to the strong dependence of plasmonic properties on nanoparticle aggregation States, the production of SERS tags with a high population of low-number aggregates is highly désirable for obtaining increased ensemble SERS responses. SERS tags with a high population of lownumber qggregates were produced either via post-synthetic sortïng techniques, or via controlled aggregation synthetic processes.

For example, field flow fractionation applied to a mixture containing single nanoparticles and nanoparticle aggregates from dimers to octamers resulted in the production of a fraction enriched with dimer (10%), trimer (21%) and tetramer (13%) SERS tags, but which however contained a high percentage of single nanoparticles (52%) (J. Am. Chem. Soc. 2010, 132, 1090310910). A mixture containing single nanoparticles and aggregates froni dimers to dodecamers was enriched in dimer (52%) and trimer (32%) SERS tags by using a centrifugal post-synthetic sorting method în a high viscosity density gradient medium, such as aqueous iodixanol density gradient medium (US9802818B2). Besides being time-consuming and expensive, the postsynthetic sorting techniques require the use of harsh conditions (for e.g. high viscosity reagents) that lead to destabîlization of uncoated SERS tags. Thus, post-synthetic sorting techniques are compatible only with silica- or polymer-coated SERS tags. Therefore, controlled aggregation synthetic processes are of great advantage for the production of SERS tags with narrow size distribution and high ratio of low-number aggregates.

Solid support assîsted aggregation allowed the production of SERS tags with a narrow size distribution. Ruan et al. (Adv. Optical Mater. 2014, 2, 65 - 73) described the synthesis of asymmetric core-satellites SERS tags having a number of 18±2 Au nanosphere satellites of 24 nm average diameter per Au nanosphere core of 180 nm average diameter. The synthesis involves the adsorption of cetyltrimethylainm onium bromide stabîlized Au nanoparticle cores on a indium tin oxide coated glass slide or a Silicon wafer, followed by immersion of the functionalized solid support in a solution of 4-aminothiophenol in water/acctonitriie to adsorb the Raman active reporter molécule on the surface of the Au nanoparticles, and subséquent immersion for 1 hour in a suspension of Au nanoparticles with an average diameter of 24 nm. The aggregation state ofthe SERS tags can be controlled by controlling the immersion time of the functionalized solid support in the suspension of Au nanoparticle satellites and/or decreasing the concentration of Au nanoparticle satellites in said suspension. The method described by Ruan et al. relies upon the use of cetyltrimethylammonium bromide surfactant for stabilizing the Au colloid, which decreases significantly the surface available for the adsorption of the Raman active reporter molécule on the Au nanoparticle core, and thereby the intensity of the SERS signal provided by the SERS tags. To tether the Au nanoparticle satellites to the Au nanoparticle core, the aggregation method employs Raman active reporter molécules presenting two functionalities with affînity for the Au surface. Hence, the method is applicable only for production of SERS tags having Raman active reporter molécules presenting two functionalities with affînity for the Au surface, which represents a high limitation in ternis of Raman active reporter molécules to be used as SERS tag fingerprint. Moreover, said method in volves long reaction times and is not suitable for tlie expédient production of high quantities of SERS tags.

A further solid support assisted assembly method of SERS tags of narrow size distribution was described by Yoon et al. (ACSNano 2012, 8, 7199 - 7208). The method is based on the sizedependent desorption propensities of Au nanoparticles adsorbed on amino-functionalized glass si ides and the use ol' alkanedithiols for tetherîng the Au nanoparticle satellites to the Au nanoparticle core. SERS tags having 13±3 Au nanoparticle satellites of 13 mn average diameter per Au nanoparticle core of 51 nm average diameter were produced. The method developed by Yoon et al. appears to enable the incorporation of any Raman active reporter molécule in the SERS tag. However, said method présents limitations in terms of sizes of Au nanoparticle that can be used and surface on the Au nanoparticle core accessible to the Raman active reporter molécules. Moreover, the method requîres long reaction times and is not suitable for the expédient production of high quantifies of SERS tags.

Thus, a need remains for reproducible, cost-efficient and versatile methods of manufacturing of SERS tags, which enable the production in an expédient manner of high quantities of SERS tags characterized by anarrow size distribution and a high ratio of low-number aggregatés. SERS tags with a high population of low-number aggregates are highly désirable for obtaining increased ensemble SERS responses.

SUMMARY OF THE INVENTION

Accordingly, it is the object ofthe présent invention to pro vide a versatile, cost-efficient and reproducible method of manufacturing SERS tags that enables the production in an expédient manner of high quantities of SERS tags characterized by a narrow size distribution and a high ratio of low-number aggregates. This is achieved by a method for manufacturing surface enhanced Raman spectroscopy (SERS) tags, preferably SERS tags for use as a security element, comprising the steps:

a) providing a first colloîd consisting essentially of nanoparticles having a plasmonic surface and substantialiy same size dispersed in an aqueous solvent, and a stabilizing agent adsorbed on the surface of said nanoparticles, and having a ζ-potential value lower than or equal to -25 mV;

b) providing a second colloid consisting essentially of nanoparticles having a plasmonic surface and substantialiy same size dispersed in an aqueous -solvent, Raman active reporter molécules adsorbed on the surface of said nanoparticles, and a stabilizing agent adsorbed on the surface of said nanoparticles, and having a ζ-potentîal value lower than or equal to -25 mV;

c) combining the first colloid with the second colloid so that the ratio between the number of nanoparticles of the first colloid and the number of nanoparticles of the second colloid is of between about 25 : 1 to about 1 : 1, preferably from about 5 : 1 to about 1 : 1, more preferably from 4 : I to about 3 : 1 to provide a third colloid;

d) inducing aggregation of the nanoparticles by any ofthe steps dl) - d3) or a combination thereof:

dl) mixing the third colloid obtained at step c) at a pH comprised between about

2.2 and the lowest pH value at which the Raman active reporter molécules hâve a net electrical charge of between 0 and 0.3;

d2) addition of a sait solution, preferably an inorganic sait solution, to the third colloid obtained at step c);

d3) addition of a water-miscible solvent to the third colloid obtained at step c); and e) stopping aggregation.

Preferably, step b) in the method claimed and described herein comprises the following steps conducted in the order bl) to b3):

bl) providing a colloid consisting essentially of nanoparticles having a plasmonic surface and substantially same size dispersed in an aqueous solvent, and a stabilizing agent adsorbed on the surface of said nanoparticles, and having a ζ-potential value lower than or equal to -25 mV;

b2) adjusting the pH of the colloid at the lowest pH value at which the Raman active reporter molécules to be adsorbed on the surface of the nanoparticles carry no net electrieal charge while maintaining the ζ-potential value lower than or equal to -25 mV, preferably lower than -40 mV; and b3) adding a solution of the Raman active reporter molécules in a solvent to the colloid obtained at step b2) while maintaining the ζ-potentîal value lower than or equal to -25 mV.

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

As illustrated by the examples El - E14, the method claimed herein allows the intégration of a variety of Raman active reporter molécules in the SERS tag and is not limited to combination of spécifie sizes of nanoparticles, enabling also the synthesis of SERS tags comprising nanoparticles having the same size as attested for e.g. by examples El - E8, and E12 - E14. Further, the manufacturing method claimed and described herein provides SERS tags with narrow size distribution as illustrated for e.g. by Fig. 3a and Fig. 3b and a high ratio of low-number aggregates as illustrated for e.g. by Fig. 3a and Fig. 3b and has significantly shorter reaction time compared to the methods known in the literature.

BRIEF DESCRIPTION OF FIGURES

Fig. la schematically represents a manufacturing method of SERS tags according to the present invention. The manufacturing method includes combining a second colloid (120a) consisting essentially of nanoparticles with a plasmonic surface and substantially same size (121a) dispersed in an aqueous solvent (not shown), wherein the nanoparticles hâve adsorbed on their surface

Raman active reporter molécules (122a) and a stabilizing agent (not shown) with a first colloid consisting essentially of nanoparticles with a plasmonic surface and substantially same size (130a) dispersed in an aqueous solvent (not shown), wherein the nanoparticles hâve adsorbed on their surface a stabilizing agent (not shown), and înducing aggregation of the nanoparticles to provide SERS tags (140a).

Fig. 1b schematically represents an embodiment of the manufacturing method of SERS tags according to the présent invention. In this spécifie manufacturing method, a continuous flow reactor comprising two pressurized tanks (111b, 112b), a tee fitting (113b) and a collecting réservoir (114b), wherein each of the two pressurized tanks (111b, 112b) is connected via a tubing to the tee fitting (113b), which itself is coimected via a tubing to the collecting réservoir (114b). The manufacturing method includes combîning and mixing simultaneously at a pH comprised between about 2.2 and the lowest pH value at which the Raman active reporter molécules hâve a net electrical charge of between 0 and 0.3 in the tubing connecting the tee fitting (113b) to the collecting réservoir (114b) of the continuous flow reactor a first colloid provided by the pressurized tank (112b) and consisting essentially of nanoparticles with a plasmonic surface and substantially saine size (130b) dispersed in an aqueous solvent (not shown), wherein said nanoparticles hâve adsorbed on their surface a stabilizing agent (not shown), with a second colloid (i20b) provided by the pressurized tank (111b) and consisting essentially of nanoparticles with a plasmonic surface and substantially same size (121b) dispersed in an aqueous solvent (not shown), wherein said nanoparticles hâve adsorbed on their surface Raman active reporter molécules (122b) and a stabilizing agent (not shown). The mixing at a pH comprised between about 2.2 and the lowest pH value at which the Raman active reporter molécules hâve a net electrical charge of between 0 and 0.3 induces nanoparticles aggregation. The aggregation step is stopped in the collecting réservoir (114b), thereby providing the target SERS tags (140b).

Fig. le schematically represents an embodiment of the manufacturing method of SERS tags according to the présent invention. In this spécifie manufacturing method, a continuons flow reactor comprises three pressurized tanks (111c, 112c, 150c), a tee fitting (113c) and a collecting réservoir (114c). Each of the two pressurized tanks (11 le, 112e) is connected via a tubing to the tee fitting (113c), which itself is connected via a tubing to the collecting réservoir (114c). The manufacturing method includes combîning and mixing simultaneously at a pH comprised between about 2.2 and the lowest pH value at which the Raman active reporter molécules hâve a net electrical charge of between 0 and 0.3 in the tubing connecting the tee fitting (113c) to the collecting réservoir (114c) ofthe continuons flow reactor a first colloid provided b y the pressurized tank (112c) and consisting essentially of nanoparticles with a plasmonic surface and substantially same size (130c) dispersed in an aqueous solvent (not shown), wherein the nanoparticles hâve adsorbed on their surface a stabilizing agent (not shown), with a second colloid (120c) provided by the pressurized tank (111c) and consisting essentially of nanoparticles with a plasmonic surface and substantially same size (121c) disp ers ed in an aqueous solvent (not shown), wherein said nanoparticles hâve adsorbed on their surface Raman active reporter molécules (122c) and a 5 stabilizing agent (not shown). The mixing at a pH comprised between about 2.2 and the lowest pH value at which the Raman active reporter molécules hâve a net electrical charge of between 0 and 0.3 induces nanoparticles aggregation. The aggregation step is stopped by introducing water stored in the pressurized tank (150c) into the tubing leading to the collecting réservoir (114c) so as to dilute the colloid, by introducing a basic solution stored in the pressurized tank (150c) into the 10 tubing leading to the collecting réservoir (114c), by introducing a polymer stored in the pressurized tank (150c) into the tubing leading to the collecting réservoir (114c), or by introducing a dielectric material precursor stored in the pressurized tank (150c) into the tubing leading to the collecting réservoir (114c), thereby providing the target SERS tags (140c).

Fig. 2a-c illustrate the variation of SERS signal intensity with the aggregation reaction time: Fig. 15 2a shows the variation of the SERS intensity with the aggregation reaction time for SERS tags manufactured as described in Example El; Fig. 2b shows the variation ofthe SERS intensity with the aggregation reaction time for SERS tags manufactured as described in Example E14; Fig. 2c shows the variation of the SERS intensity with the aggregation reaction time for SERS tags manufactured as described in Example E13. The horizontal axis corresponds to the aggregation 20 reaction time in seconds, and the vertical axis corresponds to the SERS response with 785 nm excitation. The reported SERS intensity was measured with 100 ms intégration time every 1 second at the given gold concentration, and this signal intensity has been correlated to samples diluted to 12.5 pg Au/mL and scanned at 1 second intégration.

Fig. 3a-b présent SEM images taken of SERS tags containing nanoparticles of different sizes 25 manufactured according to Examples E9 (Fig. 3a) and 10 (Fig. 3b). As attested by the SEM images, the SERS tags manufactured according to the présent invention are enriched in lownumber aggregates, such as dîmers, trimers and tetramers, and présent a narrow size distribution. Fig. 4 is a plot iliustrating the ζ-potential value of first colloid A2 (plotted as solid circles) and second colloids D2 (plotted as solid triangles), D5 (plotted as solid diamonds) and D6 (plotted as 30 solid squares), when said colloids are tîtrated with an aqueous solution of IN HCl or an aqueous solution of 1 N NaOH. The horizontal axis corresponds to the pH value, and the vertical axis corresponds to the ζ- potential value (mV), The conditions in which aggregation was observed (i.e. the colloids are unstable) are circled. As attested by Fig. 4 a variety of colloids characterized by a ζ- potential value lower than -25 mV are stable. Such colloids can be used as first and second 35 colloids, respectively in the manufacturing method according to the présent invention.

Fig. 5 illustrâtes the development of the SERS signal intensities of colloids aggregating in a flow system. Each measurement was taken at 1 meter markings along a 7 m long, transparent FEP tube. The data represented by diamonds connected by a solid line were taken on tire SERS tags produced as described by Exampie E8. The data represented by circles connected by a dotted line were taken on the SERS tags produced as described by Example Eli.

DETAILED DESCRIPTION

Définitions

The following définitions are to be used to interpret the meaning of the terms discussed in the description and recited in the daims.

As used herein, the article a/an” indicates one as well as more than one, and does not necessarily limit its referent noun to the singular.

As used herein, the term “at least ' is meant to defme one or more than one, for example one or two or three.

The terni “comprising” as used herein is intended to be non-exclusive and open-ended. Thus, for instance a solution comprising a compound A may include other compounds besides A. However, the tenu “comprising” also covers, as a particular embodiment thereof, the more restrictive meanings of “consisting essentially of and “consisting of, so that for instance “a solution comprising A, B and optionally C” may also (essentially) consists of A and B, or (essentially) consists of A, B and C.

Where the présent description refers to “preferred” embodiments/features, combinations of these “preferred'’ embodiments/features shah also be deemed as disclosed as long as this combination of “preferred’ embodiments/features is technically meaningful.

As used herein, the tenu “about” means that the amount or value in question may be the » spécifie value designated or some other value in its neighborhood. Generally, the term “about” denoting a certain value is intended to dénoté a range within ± 5% of the value. As one example, the phrase “about 100” dénotés a range of 100 ± 5, i.e. the range from 95 to 105. Preferably, the range denoted by the term “about” dénotés a range within ± 3% of the value, more preferably ± 1 %. Generally, when the term “about” is used, it can be expected that similar results or effects according to the invention can be obtained within a range of ±5% of the indicated value.

Surprisingly it was found that a method for manufacturing surface enhanced Raman spectroscopy (SERS) tags, preferably SERS tags for use as a security element, comprising the steps:

a) providing a first colloid consisting essentially of nanoparticles having a plasmonic surface and substantially same size dispersed în an aqueous solvent, and a stabilizing agent adsorbed on the surface of said nanoparticles, and having a ζ-potential value lower than or equal to-25mV;

b) providing a second colloid consisting essentially of nanoparticles having a plasmonic surface and substantially same size dispersed in an aqueous solvent, Raman active reporter molécules adsorbçd on the surface of said nanoparticles, and a stabilizing agent adsorbed on the surface of said nanoparticles, and having a ζ-potential value lower than or equal to -25 mV;

c) combining the first colloid with the second colloid so that the ratio between the number of nanoparticles of the first colloid and the number of nanoparticles of the second colloid is of between about 25 : 1 to about 1:1, preferably from about 5 : I to about 1:1, more preferably from about 4 : 1 to about 3 : 1 to provide a third colloid;

d) înducing aggregation of the nanoparticles by any of the steps dl) — d3) or a combination thereof:

dl) mixing the third colloid obtained at step c) at a pH comprised between about 2.2 and the lowest pH value at which the Raman active reporter molécules hâve a net electrical charge of between 0 and 0.3;

d3) addition ofa water-miscible solvent to the third colloid obtained at step c); and

e) stopping aggregation;

provides in a cost-efficient and expédient manner high quantifies of SERS tags with narrow size distribution and a high ratio of low-number aggregates. The method does not hâve any limitation in ternis of Raman active reporter molécule to be used as fingerprint of the SERS tag or size ofthe nanoparticles having a plasmonic surface contained in the first and second colloid, thereby enabling the manufacturing of a variety of SERS tags.

As well known to the skilled person and used herein, a SERS tag comprises one aggregate of nanoparticles presenting a plasmonic surface and Raman active reporter molécules adsorbed on the surface of the nanoparticles. The nanoparticles presenting a plasmonic surface are responsible for the génération of the electric field rcquired for the Raman amplification, while the Raman active reporter molécules provide the unique vibrational fingerprint of the SERS tag. A SERS tag may further comprise an extemal eoating layer isolatîng the nanoparticles aggregate having adsorbed on the surface Raman active molécules from the external medium. Thus, the extemal eoating layer a) isolâtes the SERS tag from the external medium thereby, preventing the Raman active reporter molécules from leaching out from the SERS tag and protecting the SERS tag from contaminations of the extemal medium that may give rise to spurious peaks, b) increases the colloïdal stability of the SERS tag, and c) provides a convenient surface for further Chemical functionalization. Extemal coating Iayers include silica and polymers, such as poly(ethylene imine) (PEl), poly(styrene-alt-maleic acid) sodium sait (PSMA), poly(diallyldimethylammonium chloride) (PDADMAC),

Owing to the exhibited SERS signal, the SERS tags obtained via the manufacturing method claimed and described herein are particularly useful as a security element for protecting documents and articles against counterfeit and illégal reproduction. As used herein, the term 'security element” désignâtes an element that can be incorporated into or applied to a security document or article for the purpose of determimng its authenticity and protecting it against counterfeit and illégal reproduction. The security element may be an indicium, image, pattern or graphie element printed, coated or sprayed on a security element or article with an ink, a vamish or a coating composition containing the SERS tags obtained via the manufacturing method according to the présent invention. Altematively, the SERS tags may function as a security element when integrated in the substrate of a security document. The term security document” and “security article refers to a document or article having a value such as to render it potentially liable to attempts at counterfeiting or illégal reproduction and which is usualiy protected against counterfeit or fraud by at least one security feature. The term “security article” as used herein en compassés ali articles that shall be protected against counterfeiting and/or illégal reproduction in order to warrant their content. Examples of security documents include without limitation value documents and value commercial goods. Typical examples of value documents include without limitation banknotes, deeds, tickets, checks, vouchers, fiscal stamps, tax ‘labels, agreements and the like, identity documents such as passports, identity cards, visas, bank cards, crédit cards, transaction cards, access documents, entrance tickets and the like. Value commercial goods encompass packaging material, in particular for cosmetic articles, nutraceutical articles, phannaceutical articles, alcohols, tobacco articles, beverages or foodstuffs, electrical/electronics articles, fabrics or jewelry, i.e. articles that shall be protected against counterfeiting and/or illégal reproduction in order to warrant the content of the packaging like for instance genuine drugs. Packaging material examples include without limitation labels, such as authentication brand labels, tamper evidence labels and seals.

Within the meaning ofthe présent invention, the term “nanoparticle” is defined as a single particle having a size corresponding to the maximum physical dimension (e.g.: length, diameter, etc.) in the range from 20 ± 5 nm to 160 ± 5 nm, preferably from 40 ± 5 nm to 140 ± 5 nm. The nanoparticle used in the présent invention has a plasmonic surface i.e. the nanoparticle has an outer surface capable of enhancing the Raman scattering of a Raman active molécule. The outer surface of the nanoparticle is made of any known SERS-enhancing material. Preferably, the SERSenhancing 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 is gold (Au). The nanoparticle used in the présent invention may be solid or hollow, and preferably solid. A solid nanoparticle may be made of a single material i.e. the SERS-enhancing material of the outer surface of the nanoparticle, or of more materials i.e. the matcrial(s) of the core ofthe nanoparticle may be difterent from the SERS-enhancing material of the outer surface of the nanoparticle. A hollow nanoparticle is a nanoparticle whose core is a void space. The nanoparticle may hâve any shape capable of being produced. Preferably, the nanoparticle is a solid Au nanoparticle. Preferably, the nanoparticle has a shape selected from the group consisting of a sphere, spheroid, rod, disk, prîsm and cube, more preferably selected from a sphere and a spheroid, and even more preferably the nanoparticle has a spheroid shape.

As used herein, the wordîng ''nanoparticles having substantially same size” means that said nanoparticles hâve a size corresponding to the maximal physical dimension (e.g.: length, diameter etc.) within ±20 mn, preferably within ±10 imi of the average size determined for said nanoparticles by électron microscopie methods, such as transmission électron microscopy (TEM) or scanning électron microscopy (SEM), or where applicable by the method of Haiss and coworkers {Anal. Chem. 2007, 79,4215-4221).

The nanoparticles ofthe first colloid, second colloid and ofthe colloid provided at step bl) are dispersed in an aqueous solvent. As used herein, the term “aqueous solvent” refers to water and a mixture of water with one or more water-miscible solvents, wherein the water miscible solvent is preferably selected from the list comprising: methanol, éthanol, propanol, isopropanol, tetrahydrofuran, W-methyl-2-pynolidone (NMP), dimethyl sulfoxide (DMSO), NJSdimethylfonnamide (DMF), acetone and acetonitrilc.

The first colloid provided at step a) of the manufacturing method, the second colloid provided at step b) of the manufacturing method, as well as the colloid provided at step bl) ofthe manufacturing method do not contain polymers or surfactants for stabilization purposes. Colloîds stabilization with polymers (for e.g.: polyvinylpyrrolidone) or surfactants (for e.g. cetyltrimethylammonium bromide) is well known in the art. However, as polymers and surfactants are added to the colloîds prior to the addition of the Raman active reporter molécules, they significantly reduce the surface avaîlable on the nanoparticles for the adsorption of the Raman active reporter molécules and increase the spacing between colloid particles within an aggregate leading therefore to SERS tags exhibiting a lower SERS signal intensity. To circumvent this drawback, the first colloid, the second colloîd, as well as the colloid provided at step bl) are surfactant-free and polymer-free.

The stabilizîng agent adsorbed on the surface of the nanoparticles of the first colloid, second colloid and of the colloid provided at step bl) is preferably selected from carboxylic acids, carboxylic acid salts, phosphoric acids, phosphoric acid salts, ascorbic acid, ascorbic acid salts, and mixtures thereof. To avoîd compétition between the Raman active reporter molécules and the stabilizîng agent during adsorption of the Raman active reporter molécules on the nanoparticles surface, preferably the stabilizîng agent does not contain groups exhibiting affinity for SERSenhancing materials, in particular gold. Examples of such groups are nîtrogen-containing groups, sulfur-containing groups, ethynyl groups, cyano groups and isocyanide groups.

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

As used herein, the term “carboxylic acid salC refers to a sodium or potassium sait of a carboxylic acid.

Preferably, the stabilizîng agent is selected from carboxylic acids, carboxylic acid salts, ascorbic acid, ascorbic acid salts, and mixtures thereof. Even more preferably, the stabilizîng agent is selected from citric acid, citric acid salts, lactic acid, lactic acid salts, ascorbic acid, ascorbic acid salts, and mixtures thereof. Citric acid salts include monosodium dihydrogen citrate, disodium hydrogcn citrate, tri sodium citrate, monopotassium dihydrogen citrate, dipotassium hydrogen citrate and tripotassium citrate. Lactic acid salts include sodium lactate and potassium lactate. Ascorbic acid salts include sodium ascorbate and potassium ascorbate. In the most preferred embodiment, the stabilizîng agent is selected from citric acid, monosodium dihydrogen citrate, disodium hydrogen citrate, trisodium citrate, monopotassium dihydrogen citrate, dipotassium hydrogen citrate and tripotassium citrate, and mixtures thereof.

Advantageously, the nianufacturing method claimed and described herein enables the intégration of any Raman active reporter molécule in a SERS tag. Preferred Raman active reporter molécules include fully conjugatcd molécules comprising an aryl group substituted by one or more substituent selected from NR¹^, -SU, —=, —and —N=, preferably from -NR¹ R² and -SH, and/or an ./V-containing heteroaryl group and/or a Ô-containing heteroaryl group, wherein the residues R¹ and R² are independently of each other selected from -H and alkyl, preferably from H and Ci-C₄-alkyl.

As known to the person skilled in the art of organic chemistry, a fully conjugated molécule is a molécule having a conjugated électrons system extending over the entire molécule. A conjugated électrons system is a system of connected p orbîtals with delocalized électrons.

As well known to the skilled person “an aryl group” is a group derived from a monocyclic or polycyclic aromatic hydrocarbon compound by removal of one hydrogen atom from a ring carbon atom. Examples of aryl groups include without limitation phenyl, naphthyl, anthracenyl, phenanthryl and pyrenyl.

A A-containing heteroaromatic compound is an aromatic compound containing a sulfur heteroatom as part of the cyclic conjugated π system. As part of the cyclic conjugated π system, the S-containing heteroaromatic compound may further contain one or more nitrogen atoms. Examples of S-containing heteroaryl groups include without limitation thiophenyl, thiazolyl, isothiazolyl, thiadiazolyl, benzothiophenyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, imidazothiazolyl and imidazothiadiazolyl.

An //-containing heteroaromatic compound is an aromatic compound containing at least one nitrogen heteroatom as part of the cyclic conjugated π system. As part of the cyclic conjugated π system, the N-containing heteroaromatic compound may further contain one or more oxygen atoms. Examples of A-containing heteroaryl groups include without limitation imidazolyl, pyrazolyl, triazolyl, tetrazolyl, benzoîmidazolyl, îndazolyl, benzotriazolyl, pyridinyl, pyrimidinyl, pyridazinyl, triazinyl, quinolinyl, isoquinolinyl, diazanaphthyl, quinalozinyl, cinnolinyl, phthalazinyl, quinoxalinyl, purinyl, aza-phenanthryl, diaza-phenanthryl, aza-anthracenyl, diazaanthracenyl, aza-pyrenyl, diazapyrenyl, oxazolyl, isoxazolyl, oxadiazolyl, benzoxazolyl, benzisoxazolyl and benzoxadiazolyl.

Preferred Raman active reporter molécules include, but are not restricted to:

- a fully conjugated compound consîsting of an aryl group substituted by one or more substituents selected from —NR'R², —SH, —=, —=N and —N=, preferably from —NR^lR² and —SH, which is connected directly or via a linker —L¹— to an aryl group substituted by one or more substituents selected from the list comprising an amino group (—NH2), an TV-alkyl amino group, an A\A^Ldialkyl-amino group, a thiol group, an ethynyi group, a cyano group and an isocyanide group, a A^r-containing heteroaryl group, or a A-containing heteroaryl group, wherein the substituents R¹ and R² hâve the meanings defined herein;

the linker —L¹ — is selected from —CR⁸=CR⁹—, —N=N-—, =—, —CR^CR¹¹—0-C6H4—, ^CR^1O=CR^U—/w-CiHr— -CR^^R^—^-CâH^, —CR'^CR¹¹—0-C6H4—CR¹²=CR¹³—, — CR¹⁰=CR^h—m-C₆H4—CR¹²=CR¹³— —CR¹⁰=CR’^l—p-C₆H4—CR¹²=CR¹³—, —CRⁱ⁴=N—N=CR¹⁵—

and the substitueras R⁸ - R^1S are selected from hydrogen, alkyl, alkoxy. alkylthio, formyl, cyano, nitro, halide, hydroxycarbonyl, and alkoxycarbonyl;

- a fully conjugated compound consisting of an TV-containing heteroaryl group, which is connected directly or via a linker —-L¹— to an A'-containing heteroaryl group, or a A-contaÏning heteroaryl group, wherein the iinker —L¹— is selected from —CR⁸~CR⁹—, —N=N—, —=— .

—CR¹⁰=CRⁿ—0-C6H4— — CR^R¹¹—m-C₆H4— —CR^CR¹¹—p-CeH»— —CR^CR¹¹—û-QH4-CR¹²=CR¹³— — CR^CR¹¹— m-C6H4—CR¹²=CR¹³— — CR'^R¹¹—p-C₆H4—CR^l2=CR¹³—, — CR¹⁴=N—N=CR¹⁵—

and

the substituents R⁸ - R¹⁵ are selected from hydrogen, alkyl, alkoxy, alkylthio, formyl, cyano, nitro, halide, hydroxycarbonyl and alkoxycarbonyl;

- a fully conjugated compound consisting of a A-containing heteroaryl group, which is connected directly or via a linker —L¹— to an 5-containing heteroaryl group, wherein the linker —L¹— is selected from —CR⁸=CR⁹—, —N=N—, —=—, —CR¹⁰=CRⁿ—o-C6H4— —CR¹⁰=CRⁿ—m-C6H₄—, —CR^W=CR^J —CR'^CR¹¹—o-CâH4—CR¹²=CR^î3—, —CR^CR¹¹—m-C6H₄--CR¹²=CR^î3— —CR’^CR¹¹—p-C₆H₄—CR¹²=CR¹³—, — CR^l4=N—N=CR¹⁵—,

and the snbstituents R⁸ - R¹⁵ are selected from hydrogen, alkyl, alkoxy, alkylthio, formyl, cyano, nitro, halide, hydroxycarbonyl and alkoxycarbonyl;

and

- a fully conjugated compound consisting of an aryl group substituted by one or more, preferably at least two, substituents selected from —NR*R², —SH, —= —=N and —N=, preferably from —NR*R² and —SH, an JV-containing heteroaryl group optîonally substituted by one or more substituents selected from —N R³ R⁴, —SH, —=, —=N and —N—, or a S-containing heteroaryl group optîonally substituted by one or more substituents selected from -NR⁵R⁶, —SU, —=, —=N and —N=, connected directly to à hydrogen atom, wherein the substituents R¹ and R² hâve the meanings defined herein and the substituents R³ - R⁶are independently of each other selected from —H and alkyl, preferably from -H and Ci-C4-alkyl.

The aryl group substituted by one or more substituents selected from —NR'R², —SH, —=, —=N and —N=, preferably from —NR?R² and ·—SH, may contain one or more further substituents preferably selected from: hydroxy, alkyl, alkoxy, alkylthio, formyl, nitro, halide, hydroxycarbonyl, alkoxycarbonyl and O-containing heteroaryl group, and more preferably selected from: alkyl, alkoxy, alkylthio, halides and O-containing heteroaryl group.

The V-contaîning heteroaryl group and the S-containing heteroaryl group may contain one or more further substituents preferably selected from: amino, N-alkyl amino, AuV-dialkyl-aniino, thiol, hydroxy, alkyl, alkoxy, alkylthio, formyl, cyano, isocyanide, ethynyl, nitro, halide, hydroxycarbonyl, alkoxycarbonyl and O-containing heteroaryl group, preferably from alkyl, alkoxy, alkylthio, halides and O-containing heteroaryl group.

Examples of ('/-containing heteroaryi groups include, but are not restricted to furanyl, benzofuranyl, isobenzofriranyl, oxazolyl, isoxazolyl, benzoxazolyl and oxadiazolyl.

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

X

wherein

A¹, B¹ and C¹ are independently of each other selected from N, CR¹⁶ and CR¹⁷, with the proviso that only one of A¹, B¹ and C¹ is N;

A², B² and C² are independently of each other selected from N, CR¹⁸ and CR¹⁹, with the proviso that only one of A², B² and C² is N;.

E¹, D¹, E², D², R¹⁶, R^!7, R¹⁸ and R¹⁹ are independently of each other selected from:

hydrogen, amino, N-alkyl amino, ΛζΛ-dialkyl-amino, thiol, hydroxy, alkyl, alkoxy, alkylthîo, fonnyl, cyano, isocyanide, alkynyl, nitro, halide, hydroxycarbonyl, alkoxycarbonyl and Ocontaining heteroaryi group, preferably from hydrogen, alkyl, alkoxy, alkylthio, halides and Ocontaining heteroaryi group; and

X is a single bond or a linker —L²— selected from —CR⁸=CR⁹—, —N=N—, —=—, —

CR¹⁰=CR^lt—o-CôIE—, —CR’^R¹¹—m-C₆H₄—, —CR^^R¹¹—/^^— —CR’^R¹¹—0-C6H4—CR^I2=CR¹³—, —CR^l0=CRⁿ—W-C6H4—CR¹²=CR¹³—, —CR¹⁰=CR^h—P-C6H4—CR¹²=CR¹³—, —CR¹⁴=N—N=CR¹⁵—,

wherein R⁸ - R¹⁵ are selected from hydrogen, alkyl, alkoxy, alkylthio, formyl, cyano, nitro, halide, hy droxy carbon yl, and alkoxycarbonyl.

Preferably, the residues A¹ and A² are N in the general formula (I). More preferably, the residues A¹ and A² are N and the substituents E¹, D¹, E², D², R¹⁶, R¹⁷, R¹⁸ and R¹⁹ are hydrogen in general formula (I).

Raman active reporter molécules include without limitation: 2-mercaptopyridine; benzenethiol; mercaptobenzoic acid; 4-nitrobenzenethiol; 3,4-dîcholorobenzenethiol; 3-fluorothiophenol; 4-fluorothiophenol; 3-5-bis(trifluoromethyl)benzenethiol;

4-mercaptophenol; biphenyl-4-thioI, 7-mercapto-4-methylcoumarin, l-(4-hydroxyphenyl)-lHtetrazole-5-thiol, 2-fluorothiophenol, 2-naphthalenethiol, 4-(((3 -mercapto-5-(2-methoxyphenyl)4H-1,2,4-triazol-4-yl)imino)methyl)phenol, (2-trifluoromethyl) benzenethiol, 4-aminothiophenol, 1-naphthalenethiol, I,r,4,l’'-terphenyl-4-thiol, biphenyI-4,4'-dithiol, thiosalicylic acid, 4-(((3mercapto-5-(2-pyridinyl)-4H-l,2,4-triazol-4-yl)imino)methyI)-l,2-benzenediol, 4-(((3-mercapto

5-(2-pyridinyl)-4H-l,2,4-triazol-4-yl)imino)methyl)benzoic, 2,3,4,6-tetrafluorobenzenethiol, (5(4-methoxyphenyl)-1,3,4-oxidazole-2-thiol), ,2-di(pyridm-4-yl)ethene, 5-(pyridïn-4-yl)l,3,4-oxadiazole-2-thiol, and l,4-bis((E)-2-(pyridin-4-yl)vinyl)benzene.

The first colloid provided at step a) is characterized by a ζ-potential value lower than or equal to -25 mV. The second colloid provided at step b) is characterized by a ζ-potential value lower than or equal to -25 mV.

As used herein, the ζ-potential value of a colloid refers to the ζ-potential value measured for said colloid at a concentration of 0.05 mg nanoparticle material/mL with a Malvem Zetasizer Nano-ZS with 1 mL folded capillary cells. If required, namely for colloids having a concentration higher than 0.05 mg nanoparticle material/mL, the colloid is diluted with deionized water to achieve the concentration of 0.05 mg nanoparticle material/mL prior to the ζ-potential measurement.

Preferably, the concentration of the nanoparticle material (mg/mL) in the first colloid and in the second colloid is lower than 0.66 mg/mL, and more preferably is comprised in between about 0.05 mg/mL. and about 0.30 mg/mL, for e.g. 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.

Following combination of the first colloid with the second colloid, for example by simple addition of the second colloid to the first colloid, so that the ratio between the number of nanoparticles of the first colloid and the number of nanoparticles of the second colloid is of between about 25 : 1 to about 1:1, preferably from about 5 : 1 to about 1 : 1‘, more preferably from about 4 : 1 to about 3:1, nanoparticles aggregation is induced. The aggregation described herein consi sis of sélective aggregation of the nanoparticles of the second colloid with the nanoparticles of the first colloid i.e. there is no aggregation of the nanoparticles of the first colloid with the nanoparticles of the first colloid, and there is no or negligible aggregation of the nanoparticles of the second colloid with the nanoparticles of the second colloid.

The nanoparticles aggregation is induced by any of the steps dl) - d3) or a combination thereof:

d2) addition of a sait solution, preferably an inorganic sali solution, to the third colloid obtained at step c);

d3) addition of a water-miscible solvent to the third colloid obtained at step c).

In a preferred embodiment inducing aggregation of nanoparticles comprises step dl), namely mixing the third colloid obtained at step c) at a prl compnsed between about 2.2 and the lowest pH value at which the Raman active reporter molécules hâve a net electrical charge of between 0 and 0.3. The net electrical charge of a Raman active reporter molécule and the pH value corresponding to said net electrical charge can be predicted through online tools such as Chemicalize.com (Chemicalize. ChemAxon. http://chemicalize.eom/#/calculation). At a pH comprised between about 2.2 and the lowest pH value at which the Raman active reporter molécules hâve a net electrical charge of between 0 and 0.3, sélective aggregation of the nanoparticles of the first colloid with the nanoparticles of the second colloid occurs.

Altematively, inducing aggregation of nanoparticles comprises step d2), namely addition of a sait solution, preferably an inorganic sait solution, to the third colloid obtained at step c). Saltinduced aggregation of nanoparticles is a well-known method of inducing nanoparticles aggregation for the perso n skilled in the art of colloid chemîstry (ChemPhysChem 2018, 19, 24 28). Examples of inorganic salts to be used at step d2) of the manufacturing method as a solution, such as an aqueous solution, include but are not restricted to: sodium fluoride, sodium chloride, sodium bromide, sodium iodide, magnésium chloride, potassium chloride and mixtures thereof.

In a further alternative embodiment, inducing aggregation of nanoparticles comprises step d3), namely addition of a water-miscible solvent to the third colloid obtained at step c). The watermiscible solvent is preferably selected from the list comprising: methanol, éthanol, propanol, isopropanol, tetrahydrofuran, /V-methyl-2-pyrrolidone (NMP), dimethyl sulfbxide (DMSO), Ν,Νdimethylformamide (DMF), acetone and acetonitrilc.

A preferred manufacturing method according to the présent invention comprises the steps of:

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

b) providing a second colloid consisting essentially of nanoparticles having a plasmonic surface and substantially same size dispersed in an aqueous solvent, a Raman active reporter molécule of general formula (1) adsorbed on the surface of said nanoparticles, and a stabilizing agent adsorbed on the surface of said nanoparticles, and having a ζ-potential value lower than or equal to -25 mV;

c) combining the first colloid with the second colloid so that the ratio between the number of nanoparticles of the first colloid and the number of nanoparticles of the second colloid is of between about 25 : I to about 1:1, preferably from about 5 : 1 to about 1:1, more preferably from about 4 : 1 to about 3 :1 to provide a third colloid;

d) inducing aggregation of the nanoparticles by any of the steps dl) - d3) or a combination thereof:

dl) mixîng the third colloid obtained at step c) at a pH comprised between about 2.2 and about 6.1, preferably between about 2.6 and about 5.7;

Preferably in the manufacturing methods described herein, step b) comprises the following steps conducted in the order bl) to b3):

bl) providing a colloid comprising nanoparticles having a plasmonic surface and substantially same size dispersed in an aqueous solvent, and a stabîlizing agent adsorbed on the surface of said nanoparticles, and having a ζ-potential value lower than or equal to -25 mV;

b2) adjusting the pH of the colloid at a value higher than the lowest pH value at which the Raman active reporter molécules to be adsorbed on the surface of the nanoparticles carry no net electrical charge while maintaining the ζ-potential value lower than or equal to -25 mV, preferably lower than -40 mV ; and b3) adding a solution of the Raman active molécules in a solvent to the colloid obtained at step b2) while maintaining the ζ-potential value lower than or equal to -25 mV.

The colloid provided at step bl) of the manufacturing method claimed herein consists essentially of nanoparticles having a plasmonic surface and substantially same size dispersed in an aqueous solvent, and a stabîlizing agent adsorbed on the surface of said nanoparticles. The first colloid provided at step a) of the manufacturing method claimed herein consists essentially of nanoparticles having a plasmonic surface and substantially same size dispersed in an aqueous solvent, and a stabîlizing agent adsorbed on the surface of said nanoparticles. The second colloid provided at step b) of the manufacturing method claimed herein consists essentially of nanoparticles having a plasmonic surface and substantially same size dispersed in an aqueous solvent, Raman active reporter molécules adsorbed on the surface of said nanoparticles, and a stabîlizing agent adsorbed on the surface of said nanoparticles. Thus, the nanoparticles of the first colloid and the nanoparticles of the second colloid do not présent on their surface molécules or organisais enablîng the spécifie interaction via a lock-and-key mechanism between the nanoparticles of the first colloid and the nanoparticles of the second colloid. Examples of such molécules include antibodies, proteins, antigens, complementary DNA strands and complementary RNA strands. Examples of such organisais include bacteria, viruses and spores.

Thus, the first colloid, the second colloid, as well as the colloid provided at step bl) besides being surfactant- and polymer-free, are also free of molécules such as antibodies, proteins, antigens, complementary DNA strands and complementary RNA strands, and organisms, such as bacteria, viruses and spores.

At step b2) of the manufacturing method according to the présent invention, the pH of the colloid is adjusted at a value higher than lowest pH value at which the Raman active reporter molécules to be adsorbed on the surface of the nanoparticles carry no net electrical charge, while maintaining the ζ-potential value lower than or equal to -25 mV, preferabiy lower than -40 mV. The net electrical charge of a Raman active reporter molécule and the pH value corresponding to said net electrical charge can be predictcd through online tools such as Chemicalize.com (Chemicalize. ChemAxon. http://ehemicalize.eom/#/calculation).

Preferabiy, at step b2), the pH of the colloid is adjusted in between about 8.2 and about 12.1, while maintaining the ζ-potential value lower than or equal to -25 mV, preferabiy lower than -40 mV. In a more preferred embodiment, the pH of the colloid is adjusted at step b2) at a value of about 11.0.

At step b3) according to the présent invention, a solution of Raman active reporter molécule in a solvent is added to the colloid obtained at step b2) while maintaining the ζ-potential value lower than or equal to -25 mV. The solvent used for preparing the solution of Raman active reporter molécule encompasses any aqueous solvent and any organic solvent suitable for dissolving the Raman active reporter molécule. Examples of organic solvents include, but are not limited to alcohols, preferabiy selected from methanol, éthanol, propanol and isopropanol, tetrahydrofuran, jV-methyl-2-pyrrolidone (NMP), dimethyl sulfoxîde (DMSO), dimethylfonnamide (DMF), aceione and acctonitrile. Aqueous solvents include, but are not limited to water and mixtures of water and water-miscible solvents, such as methanol, éthanol, propanol, isopropanol, tetrahydrofuran, 7V-methyl-2-pynOlidone (NMP), dimethyl sulfoxide (DMSO), ΛζΝ-diniethylfornïamide (DMF), acetone and acctonitrile.

b) providing a second colloid consisting essentially of nanoparticles having a plasmonic surface and substantially same size dispersed in an aqueous solvent, a Raman active reporter molécule of general formula (1) adsorbed ou the surface of said nanoparticles, and a stabilizing agent adsorbed on the surface of said nanoparticles, and having a ζ-potential value lower than or equal to -25 mV;

c) combining the first colloid with the second colloid so that the ratio between the number ot nanoparticles of the first colloid and the number of nanoparticles of the second colloid is of between about 25 : 1 to about 1:1, preferably from about 5 : 1 to about 1:1, more preferably front 4 : 1 to about 3 : 1 to provide a third colloid;

d) inducing aggregation ofthe nanoparticles by any ofthe steps dl)-d3) or a combination thereof:

dl) mixing the third colloid obtained at step c) at a pH comprised between about 10 2.2 and about 6.1, preferably between about 2.6 and about 5.7;

d3) addition of a water-miscible solvent to the third colloid obtained at step c); and

e) stopping aggregation, wherein step b) comprises the following steps conducted in the order bl) to b3):

b2) adjusting the pH of the colloid at a value comprised between about 8.0 and about 12.1, preferably between about 8.2 and 12.1, while maintaining the ζ-potential value lower than or equal to -25 mV, preferably lower than -40 mV; and b3) adding a solution of a Raman active reporter molécule of general formula (I) in a solvent to the colloid obtained at step b2) while maintaining the ζ-potential value lower than or equal to -25 mV.

The inventors found that the intensity of the signal of the SERS tags manufactured according to the présent invention can be further increased by ensuring that a sub-monolayer or a monolayer of Raman active reporter molécules is adsorbed on the surface of the nanoparticles containcd by the second colloid. Thus, a further preferred embodiment according to the présent invention is directed to a manufacturing method of SERS tags as claimed herein, wherein the nanoparticles of the second colloid hâve absorbed on their surface a sub-monolayer or monolayer of Raman active reporter molécules. As used herein, a monolayer of Raman-active reporter molécules adsorbed on the surface ofthe nanoparticles of the second colloid refers to a one Raman active reporter moleeule-thick layer adsorbed on the surface of said nanoparticles. As used herein, a sub-monolayer of Raman active reporter molécules refers to an incomplète üionolayer of Raman- active reporter molécules, To ensure that a sub-monolayer or monolayer of Raman-active reporter molécules is adsorbed on the surface of the nanoparticles of the second colloid, the amount of the Raman active reporter molécules added at step b3) to the colloid obtained at step b2) has to be calculated by methods well known to the skilled person depending on the shape and size of the of the nanoparticles ofthe second colloid.

The manufacturing method claimed herein enables also the préparation of SERS tags comprising a mixture of different Raman active reporter molécules i.e. a mixture of two or more different Raman active reporter molécules. This is particularly advantageous because it enables the access to a variety of SERS tags, wherein each of said SERS tags is characterized by a unique SERS signal, by combining a limited number of different Raman active reporter molécules in different ratios. To achieve such SERS tags, the nanoparticles of the second colloid provided at step b) are prepared so that to hâve absorbed on their surface a mixture of different Raman active reporter molécules (i.e. a mixture of two or more different Raman active reporter molécules). Such second colloid can be prepared by using at step b3) of the manufacturing method a solution of Raman active molécules in a solvent comprising two or more different Raman active reporter molécules or by conducting successively step b3) of the manufacturing method and using each time a different solution containing a different Raman active reporter molécule. Thus, a further embodiment according to the présent invention is directed to a manufacturing method, wherein at step b3) the solution of the Raman active reporter molécules in a solvent comprises a mixture of two or more different Raman active reporter molécules. Other further embodiment according to the présent invention is directed to a manufacturing method, wherein step b3) is conducted successively n times with n>2, using each time a solution containing a Raman active reporter molécule, which is structurally different from the Raman active reporter molécules used in the remaîning n-1 solutions used in the remaining n~l steps. As used herein, different Raman active reporter molécules refer to Raman. active reporter molécules having a different Chemical structure and providing a different SERS spectrum.

To further increase the low-number aggregates population, and thereby the intensity of the SERS signal provided by the SERS tags manufactured by the manufacturing method claimed herein, it is preferred that at step c) the ratio between the number of nanoparticles ofthe first colloid and the number of nanoparticles of the second colloid is of between about 5 : 1 to about 1:1, preferably from about 4 : 1 te about 3 : 1. As attested for instance by exampie 10 and Fig. 3b, a ratio between the number nanoparticles ofthe first colloid and the number of nanoparticles ofthe second colloid of between about 5 : 1 and about 1 : 1 allows the access to SERS tags with high population of low-number aggregates, such as dimers, trimers and tetramers.

To induce aggregation by the method described at step dl) it is required to mix the third colloid obtained at step c) at a pH compnsed between about 2.2 and the lowest pH value at which the Raman active reporter molécules hâve a net electrical charge of between 0 and 0.3, which for a Raman active reporter molécule of general formula (I) is a pH comprised between about 2.2 and about 6.1, preferably between about 2.6 and about 5.7. This can be achieved either by addition of an acid solution to the third colloîd obtained at step c), or by adjusting the pH ofthe first colloid so that the colloid obtained at step dl) has the required pH value.

Hence, an embodiment according to the présent invention is directed to a manufacturing method wherein step dl) further comprises addition of an acid solution to the third colloid obtained at step e) while mixing so that the pH of the resulting colloid is comprised between about 2.2 and the lowest pH value at which the Raman active reporter molécules hâve a net electrical charge of between 0 and 0.3, which in the case of a Raman active reporter molécule of general formula (I) is a pH comprised between about 2.2 and about 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 nîtric acid.

To avoid an additional manufacturing step, it is convenient to adjust the pH value of the first colloid so tirât the colloid obtained at step dl) has a pH comprised between about 2.2 and the lowest pH value at which the Raman active reporter molécules hâve a nét electrical charge of between 0 and 0.3, which in the case of a Raman active reporter molécule of general formula (I) îs a pli comprised between about 2.2 and about 6.1, preferably between about 2.6 and about 5.7.

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

el) adjusting the pH of the colloid obtained at step d) at a value higher than the lowest pH value at which the Raman active reporter molécules to be adsorbed on the surface of the nanoparticles carry no net electrical charge;

e2) diluting the colloid obtained at step d) with water, preferably so that the nanoparticles concentration in the colloid is below 6*10⁹ nanoparticIcs/mL;

e3) addition of a polymer to the colloid obtained at step d);

e4) addition of a dielectric material precursor to the colloid obtained at step d).

In the inventive manufacturing method claimed herein, aggregation can be stopped by any ofthe methods el)-e4).

Adjustment of the pH of the colloid obtained at step d) at a value higher than the lowest pH value at which the Raman active reporter molécules to be adsorbed on the surface of the nanoparticles carry no net electrical charge as described by step el), results in an increase of the electrostatic repulsions between the nanoparticles aggregates, leading to the stopping of the aggregation process. The net electrical charge of a Raman active reporter molécule and the pH value corresponding to said net electrical charge can be predicted through online tools such as Chcmicalize.com (Chemicalize. ChemAxon. http://chemicalize.eom/#/calcuIation).

Addition of a polymer to the colloid obtained at step d) as described at step e3) results in the increase of steric repulsions between the nanoparticles aggregates and leads to the stopping of the aggregation process. Advantageously, the polymer used at step e3) does not influence the SERS signal exhibited by the SERS tag. Suitable polymers include but are not limited to polyvinylpyrrolidone and polyethylene glycol.

Addition of a dielectric material precursor to the colloid obtained at step d) as described at step e4) results in the encapsulation of the nanoparticles aggregates with at least one layer of dielectric material, which stabilizes the SERS tags and împlicitly results in the stopping of the aggregation process. Preferably, the dielectric material precursor is a silica precursor. Silica precursors include, but are not restricted to solutions of tetraethyl orthosilicate and 3aminopropyltrimethoxysilane in éthanol, solutions of tetraethyl orthosilicate and (3mercaptopropyl)trimethoxysilane in éthanol, solutions of sodium silicate in water, and solutions of (3-mercaptopropyl)trimethoxysilane in water.

Altematively, the aggregation process can be stopped by diluting the colloid obtained at step d) with water. Preferably, the aggregation is stopped by diluting the colloid obtained at step d) with water so that to reach a concentration of nanoparticle material [pg/mL] lower than or equal to 12.5 pg/mL (see for e.g.: El - E8, Eli - EI4). Altematively, the aggregation is stopped by diluting the colloid obtained at step d) with water so that to reach a nanoparticles concentration below 6*10⁹ nanoparti cles/mL.

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

In a further preferred embodiment, steps c) and d) of the manufacturing method claimed herein are conducted simultaneously in a continuous flow system. In this spécifie manufacturing method, a continuous flow reactor is used. Such continuous flow reactor is schematically represented in Fig. lb and Fig. le and comprises two pressurized tanks (111b, 112b, 111c, 112c) connected via a tubing to a tee fitting (113b, 113c) and a collecting réservoir (114b, 114c). The second colloid is stored in the pressurized tank (111b, 111c), while the first colloid is stored in the pressurized tank (112b, 112c). A vessel open to the atmosphère (114b, 114c) is used to collect the SERS tags and optionally to stop aggregation (1 14c). A tubing connecting each-ofthe pressurized tanks (111b, 112b, 111c, 112c) to the tee fitting (113b, 113c) is used for colloids transport. A further tubing connecting the tee fitting (113b, 113c) to the collection vessel (114b, 114c) is used for conducting aggregation. The nanoparticles aggregates obtained via aggregation are collected in the collection vessel (114b), where the aggregation is stopped.

When steps c) and d) of the manufacturing method are conducted simultaneously in a continuous flow System, it is further preferred that step e) is also conducted in a continuons flow System.

As mentioned above, the nanoparticles used in the manufacturing method of SERS tags may hâve any shape capable of being produced, such as sphere, spheroid, rod, disk, prism and cube. Preferably, the shape of the nanoparticles having a plasmonic surface used in the inventive manufacturing method claimed herein is selected from a sphere and a spheroid. Even more preferably said nanoparticles hâve a spheroid shape.

In a preferred embodiment, the nanoparticles of the first colloid and the nanoparticles of the second colloid hâve the same size. Conveniently, for such manufacturing method of SERS tags the colloid used for the préparation of the second colloid (i.e. the colloid provided at step bl)) is the first colloid.

In an alternative embodiment, the nanoparticles size of the first colloid is different from the nanoparticles size of the second colloid. For example, the size of the nanoparticles in the first colloid may be lower than the size of the nanoparticles in the second colloid resulting in SERS tags having structures similar to the SERS tags depicted by Fig. 3a, or the size ofthe nanoparticles in the first colloid may be bigger than the size of the nanoparticles in the second colloid resulting in SERS tags having structures similar to the SERS tags depicted by Fig. 3b.

The manufacturing method claimed herein is preferably conducted with colloîds, wherein the plasmonic surface of the nanoparticles in the first colloid and/or the plasmonic surface of the nanoparticles in the second colloid is made of gold, more preferably with colloîds comprising solid gold nanoparticles, and even more preferably with citrate stabilized gold colloîds.

EXAMPLES

The présent invention is now described in greater detail with respect to the following non-limiting examples.

General

The following reagents were obtained from the following suppliers:

Gold chloroaurate trihydrate (>99.9%; CAS no.: 16961-25-4), sodium borohydride (99.99%; CAS no.: 16940-66-2), sodium citrate tribasic dihydrate (>99.5%; CAS No.: 6132-04-3), (£)-1,2 di(pyridin-4-yl)ethene (97%; CAS No.: 13362-78-2), 5-(pyridin-4-yl)-l,3,4-oxadiazole-2-thiol (97%; CAS no.: 15264-63-8), sodium hydroxide (ACS reagent, >97%; CAS No.: 1310-73-2), hydroxylamine hydrochloride (99.999%; CAS No.: 5470-11-1), terephthalaldehyde (ReagentPlus, 99%, CAS No.: 623-27-8), 4-methylpyridine (99%; CAS No.: 108-89-4), acetic anhydride (ReagentPlus, >99%; CAS No.: 108-24-7), dichloromethane (anhydrous, 99.8%; CAS No.; 75-092), and methanol (HPLC, >99.9%; CAS No.: 67-56-1) were purchased from Sigma Aldrich. Hydrochloric acid (trace métal grade, 34-37%; CAS No.: 7647-01-0) was purchased from Fisher Scientific.

The ζ-potential values were measured using a Malvem Zetasizer Nano-ZS with I mL folded capillary cells (DTS1060). Optical absorption spectra were recorded on an Agilent 8453 spectrophotometer and on a Perkin Elmer Lambda 650.

Scanning électron microscope (SEM) images were taken on a Hitachi S-4500.

785 nm Raman spectra were obtained on Océan Optics QE 6500.

The nominal 140 nm gold colloid particles were sized by sending a sample to EAG Laboratories for transmission électron microscopy (TEM) imaging. The images were analyzed with ImageJ software (https://imagej.nih.gov/ij/). A batch was considered to be nominally 140 nm, if the number average particle size was within ±9 nm of the nominal diameter, wherein the number average particle size was determined by the measurement of 230 individual particles in TEM micrograpbs. The size-related characteristic selected for describing the individual particles was the circle équivalent (CE) diameter, which corresponds to the diameter of a circle that would hâve the same area as an orthographie projection of the particle.

The gold nanoparticle diameter d (nm) for the 40, 60, 90 nm nanoparticle batches were calculated by the method oÎIlaiss and coworkers (Anal. Chem. 2007, 79, 4215-4221) using the équation

0.0216 nm where T^is the surface plasmon résonance peak position in the extinction plot taken ofthe colloid sample on a Perkin Elmer Lambda 650 UV Vis. A batch was considered to be nominally 40 nm, 60 nm or 90 nm, if the surface plasmon résonance peak position correlated to a diameter within ±9 nm of the nominal diameter.

I. Préparation of Au colloid stock solution (SI - S4).

The gold colloid stock solutions (SI - S4) characterized by the Au nanoparticle size (nm), the Au concentrations (mg/mL) and the pH values indicated în Table 1 were manufactured as described below:

1.1 Préparation of 40 nm gold colloid stock solution (Si)

In a jacketed thoroughly cleaned 100 L glass reactor (ChemGlass), 79.5 1 of 17 ΜΩ water was chilled to 3.5±0.5 °C. Gold chloroaurate trihydrate (100g) as a20 wt% solution in water was added while stirring with an impeller at 400 rpm. Sodium citrate tribasic dihydrate (174.3 g) as a 30 wt% solution in water and hydroxylamine hydrochloride (155.6 g) as a 23.5 wt% solution in water were combined and added to the reactor. After 10 s, 800 pL of sodium borohydride as a 0.063 wt% solution in 0.01 N sodium hydroxide 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 colloid sto.ck solution (SI) having a gold concentration of 0.25 mg Au/mL and a pH value of about 2.4.

1.2 Préparation of 60 nm gold colloid stock solution (S2)

In a jacketed thoroughly cleaned 100 L glass reactor (ChemGlass), 79.5 1 of 17 ΜΩ water was chilled to 3,5±0.5 °C. Gold chloroaurate trihydrate (200g) as a 20 wt% solution in water was added while stirring with an impeller at 400 rpm. Sodium citrate tribasic dihydrate (173.3 g) as a 30 wt% solution in water and hydroxylamine hydrochloride (217.1 g) as a 17.5 wt% solution in water were combined and added to the reactor. After 10 s, 800 pL of sodium borohydride as a 0.052 wt% solution în 0.01 N sodium hydroxide 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 with 17 ΜΩ water to 160 L to give a 60 nm Au colloid stock solution (S2) having a gold concentration of 0.25 mg Au/ml and a pli of about 2.4.

1.3 Préparation of 90 nm gold colloid stock solution (S3)

In a jacketed thoroughly cleaned 100 L glass reactor (ChemGlass), 79.5 1 of 17 ΜΩ water was chilled to 3.5+0.5 C. Gold chloroaurate trihydrate (200g) as a 20 wt% solution in water was added while ' stirring with an impeller at 400 rpm. Sodium citrate tribasic dihydrate (173.3 g) as a 30 wt% solution in water and hydroxylamine hydrochloride (217.1 g) as a 17.5 wt% solution in water were combined and added to the reactor. After 10 s, 900 pl of sodium borohydride as a 0.01 wt% solution in 0.01 N sodium hydroxide was injected into the reactor. The reagents were allowcd to react for 2 minutes, and then drained into a clean drum. The batch was diluted with 17 ΜΩ water to 160 L to give a 90 nm Au colloid stock solution (S3) having a gold concentration of 0.25 mg Au/mL and a pH of about 2.4.

1.4 Préparation of 140 nm gold colloid stock solution (S4)

In a 2 L glass jug, 1.5 1 of 17 ΜΩ water was stirred at room température. Gold chloroaurate trihydrate (2.5 g) as a 20 wt% solution in water was added while stirring. Sodium citrate tribasic dihydrate (15.37 g) as a 30.7 wt% solution in water and hydroxylamine hydrochloride (8.37 g) as a 16.7 wt% solution in water were combined and added to the reactor. After 5 s, 25 pL of sodium borohydride as a 0.01 wt% solution in 0.01 N sodium hydroxide was injected into the reactor. Additional stirring for 15 minutes provided a 140 nm Au colloid stock solution (S4) having a gold concentration of 0.25 mg Au/mL and a pH of about 2.4.

Table 1: Characterization ofthe Au colloid stock solutions (SI - S4).

Au colloid stock solution no.:	Au nanoparticles size (nm)	Au concentration (mg/mL)	pH
SI	40	0.25	«2.4
S2	60	0.25	«2.4
S3	90	0.25	«2.4
S4	140	0.25	«2.4

H- Préparation of a first colloid (Al — A9) (step a) of the manufacturing method)

First colloids (Al - A9) were obtained starting from Au colloid solutions SI, S2 and S4. If necessary (for e.g.: Au colloids Al and A7) the Au colloid stock solutions were diluted with deionized water so that to obtain the Au concentration (mg/mL) indicated in Table 2. Further, if necessary (for e.g.: Au colloids A3, A4, A5, A6, A8, A9), the Au colloid stock solutions per se or after dilution are treated with an aqueous 0.1 mM solution of NaOH or an aqueous 0.1 mM solution of HCl so that to adjust the pH value of the first colloids to the pH value indicated by Table 2.

Table 2: Characterization of the first colloids (Al - A9).

Au colloid stock solution no.:

Au nanoparticles size (nM)

Au concentration (mg/mL) pH value

Al	60	0.10	«2.5
A2	60	0.25	«2.4
A3	60	0.25	«3.0
A4	60	0.25	«1.7
A5	60	0.25	«8.8
A6	60	0.25	«4.2
A7	140	0.10	«2.4
A8	40	0.25	«4.2
A9	60	0.25	«6.0

Only stable first colloids i.e. colloids where no aggregation occurs are suitable to be used in the manufacturing method according to the présent invention. First colloids Al - A9 are stable i.e. no aggregation of the Au nanoparticles could be identified by visual détection of a change of color from pink to purple. Further, as attested by Figure 4, a variety of Au colloids characterized by a zeta-potential value lower than or equal to

-25 mV measured as described at item IV below are stable. Therefore, such Au colloids are also useful as first colloids in the manufacturing method according to the présent invention.

1Π. Préparation of a second colloid containing Au nanoparticles and Raman active reporter molécules adsorbed on the surface of Au nanoparticles (Dl - DU) (step b) of the manufacturing method)

Au colloids (Bl - B8) characterized by the Au concentrations (mg/mL) and the pH values indicated in Table 3 were prepared starting from the Au colloid stock solutions SI - S3. The préparation involves, ifnecessaiy (for e.g.: Au colloids Bl and B5) dilution ofthe Au colloid stock solutions with deionized water so that to obtain the-indicated Au concentration (mg/mL), and adjustment of the pH value by addition of an aqueous 1 M NaOH solution to the Au colloid stock solutions per se or aller dilution.

Table 3: Characterization of Au colloids Bl - B9.

Au colloid no.:	Au nanoparticles size (nM)	Au concentration (mg/inL)	pi 1 value
Bl	60	0.10	«11.2

B2	60	0.25	«11.2
B3	60	0.25	«9.8
B4	60	0.25	«12.1
B5	90	0.10	«1 L0
B6	60	0.25	«7.1
B7	60	0.25	«5.1
B8	40	0.25	«11.0
B9	60	0.25	«8.2

The Raman active reporter molécule 1,4-bis((£)-2-(pyridm-4-yl)vmyl)benzene was synthesized as described below:

In a 50 ml round bottom flask with a stir bar was charged terephthalaldehyde (3.44 g, 25.7 mmol), 4-methylpyridine (9.57 g, 103 mmol), and acetic anhydride (25 mL). The mixture was refluxed until no more aldéhyde was present (4 h, checked by TLC). The reaction was cooled to room température and quenched by pouring it into 100 ml ice water. The cold mixture was neutralized to pH 7 using an aqueous 6 N solution of NaOH and the resulting brown precipitate was filtered, washed with water and air dried. Extraction with dichloromethane, followed by solvent concentration to dryness provided the crude product that was purifïed by flash column chromatography on silica gel (methanol/dichloromethane: 5/95) to afford 0.875 g of the target Raman active reporter molécule as a yellow solid (12%).

The second colloid (DI — DU) containing Au nanoparticles having adsorbed on their surface Raman active reporter molécules was prepared by adding a volume of 0.1 mM Raman active reporter molécule solution in éthanol to 20 mL Au colloid (B1 - B5, B9) or 3L Au colloid (B8), tbllowed by stining ofthe resulting mixture for 30 min (second colloid DI - D7, D9 — Dll)/1 h (second colloid D8) at room température. Table 4 provides a summary of the Raman active reporter molécules and volumes of Raman active reporter molécule solution in éthanol used for the préparation of the second colloid DI — DI 1.

Table 4: Préparation ofthe second colloids DI — DI 1.

Second colloid no.:	Starting Au	Raman active reporter	Volume 0.1 mM Raman
	colloid	molécule	active reporter molécule solution

DI	B1	(£)-1,2-di(pyridin-4-yl)ethene	300 pL
D2	B2	(£)-1,2-di(pyridin~4-yl)ethene	750 pL
D3	B3	(£)-1,2-di(pyridin-4-yl)ethene	750 pL
D4	B4	(£)-l ,2-di(pyridin-4-yl)ethene	750 pL
D5	B2	5-(pyridin-4-yl)-1,3,4oxadiazole-2-thiol	750 pL
D6	B2	1,4-bis((£)-2-(pyridm-4yljvinyljbenzene	750 pL
D7	B5	(E)-1,2-di(pyridin-4-yl)ethene	240 pL
D8	B8	(E)~ 1,2-di(pyridin-4-yl)ethene	127.5 mL
D9	B9	(£)-l ,2-di(pyridin-4-yl)ethene	750 pL
D10	B1	1,4-bîs((£)-2-(pyridin-4yljvinyl) benzene	300 pL
DU	B1	5-(pyridin-4-yl)·-1,3,4oxadiazoIe-2-thiol	300 pL

Treatinent of 20 ml of the Au colloids B6 and B7 having a pH value lower than the lowest pH value at which (£)-1,2-di(pyridîn-4-yl)ethene carry no net electrical charge as predicted by Chemicalize.com with 750pL of a 0.1 mM solution of (£)-l,2-di(pyridin-4-yi)ethene in éthanol, 5 followed by stirring of the resulting mixture for 30 min at room température resulted in unstable colloids as indicated by visual détection of a color change from pink to purple of the mixture during stirring. Unstable colloids cannot be used in the manufacturing method according to the présent invention. To ensure the stability of the second colloid i.e. to avoid aggregation of the Au nanoparticles contained in said colloid, it is important that during préparation and storage of said 10 colloid the ζ -potential value is lower than or equal to —25 mV and the pH value is higher than the lowest pH value at which the Raman active reporter molécule to be adsorbed on the Au nanoparticles carry no net electrical charge, wherein said value can be predicted for example with Chemicalize.com. In this sense, the pH value of the Au colloid (B1 - B5, B8, B9) used for the préparation of the second colloid is adjusted before treatment with the Raman active reporter 15 molécule to a value higher than the lowest pH value at which the Raman active reporter molécule to be adsorbed on the Au nanoparticles contained by the Au colloid (B1 - B5, B8, B9) carry no net electrical charge, wherein said value can be predicted for example with Chemicalize.com.

IV. Stability of the first and second colloids. To be suitable for use in the manufacturing method according to the présent invention providing SERS tags with high population of low-size aggregates, the first and second colloids must be stable i.e. during préparation and storing of said colloids, nanoparticles aggregation ofthe nanoparticles must be avoided. This can be ensured by maintaining the ζ -potential value of said colloids at a value lower than or equal to -25 mV. As shown by Fig. 4 and Table 5, a variety of Au colloids can be used as first and second colloids in the manufaeturing method of SERS tags according to the présent invention.

The ζ-potcntial of the first colloid A2 and second colloids D2, D5 and D6 was measured as a function of pH at a concentration of 0.05 mg Au/mL at room température. The results are reported în Table 5 and plotted in Fig. 4.

The ζ-potential measurements were made on a Malvem Zetasizer Nano-ZS with I mL folded capillary cells. The physical properties of water at 25 °C and gold were preloaded on the instrument First colloid A2 and second colloids D2, D5 and D6 were each diluted with deionized water to a concentration of 0.05 mg Au/mL.

The ζ -potential values of the first colloid A2 and of the second colloid D2, D5 and D6 at about the pH used for the SERS tags synthesîs according to the invention are reported in the 2^Ild column of Table 5. The ζ -potential values ofthe first colloid A2 and of the second colloid D2, D5 and D6 at the pH at which the colloids become unstable are reported in the 3^rd column of Table 5. Instabîlity of the colloid was determined by visual détection of a color change of the colloid from pink to purple.

Table 5: ζ -potential measurements of first and second colloids.

Au-colloid no. diluted at 0.05 mg Au/mL [Au-concentration]	ζ -potential after dilution (pH after dilution)	ζ -potential at which the Au colloid becomes unstable (pH at which the Au colloid becomes unstable)
A2	-43 mV (at pH ~ 2.5)	-17 mV (atpH« 1.6)
D2	-44 mV (at pH rî 10)	-22 mV (at pH rî 6)
D5	-38 mV (at pH« 10)	-16 mV (at pH « 3.4)
D6	-36 mV	-23 mV

(at pH« 10) (at pH « 7)

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

V. l SERS tags containing Au nanoparticles having the same size (Examples El — E8, E12 - E14) V.l.a Batch aggregation (Examples El - E7, E12 - E14)

The second colloid (Dl - D6, D9 - DU, 20 mL) was quickly poured to S0 mL of the first colloid (Al — A5, A9) and the mixture was stirred in a mîxing réservoir with a magnetic stirrer bar. 30 sec after colloîds combination, a 125 pL aliquot was sampled and dîluted to 1 mL with water, thereby stopping aggregation. At this dilution (12,5 pg Au/mL corresponding to about 5.72*10⁹ Au nanoparticles/mL) aggregation ceased, and the SERS signal was measured using 785 nm laser excitation and a QE65000 spectrometer purchased from Océan Optîcs Inc. set to one second intégration time. The results of the SERS signal measurements conducted on the SERS tags manufactured as described above are depicted in Table 6.

As attested by the examples El - E7, and E12 - E14 according to the inventive method claimed herein, and the comparative examples Cl - C3, sélective aggregation of the parti ci es of the first colloid and the particles ofthe second colloid does not occur at low pH value such as 1.8 and at pH values higher than the lowest pH value at which the Raman active reporters molécules hâve a net electrical charge of between 0 and 0.3, as predicted by Chemicalize.com.

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Table 6: SERS signal intensity exhibited by the SERS tags according to Examples El - E7, E12 and comparative examples Cl - C3.

V.l.b Continuons flow system aggregation (Example E8)

A schematic of the continuons flow reactor used in the manufacturing method according to the présent invention is presented in Fig. 1b. A pressurized tank (111b) holds the second colloid, and a second pressurized tank (112b) holds the first colloid. A vessel opened to the atmosphère (114b) is used to collect the SERS tags and contains a concentrated polymer solution for stopping the aggregation reaction. Transparent fluorinated ethylene propylene (FEP) from Cole P armer® Scientifïc Experts tubing with mm nominal internai diameter is used for colloid transport. 30 cm of the.FEP tubing was used to connect the pressurized tank (111b) to the T-junction (113b). 30 cm of the FEP tubing was used to connect the pressurized tank (112b) to the T-junction (113b). 7 m of FEP tubing was used to connect the T-junction (113b) to the collection vessel (114b). This section of tubing between the T-junction (113b) and the collection vessel (114b) is the section in which aggregation occurs.

Colloids A 8 (12 L) and D8 (3 L) described above were placed in tanks 112b and 111b, respectively. The tank (11 Ib) containing second colloid (D8) was pressurized such that the colloid flowrate was 0.66 L/min. The tank (112b) containing the first colloid (A8) was pressurized such that the colloid flowrate was 2.65 L/min. After combining at the T-junction, the combined colloid, characterized by a ratio between the number of nanoparticles of the first colloid A8 and the number of nanoparticles of the second coiioid D8 is of «4:1, has a pH value of about 4.8 and a flow rate in the aggregation tubing of 3.31 L/min, giving a linear velocity of J. 1 m/s. SERS measurements were taken tlirough the transparent FEP tubing at 1 m markings down the length of the aggregation tubing with a probe coupled with a fiber optic cable to a Océan Optîcs QE65000 spectrometer with 785 nm excitation. The intégration time was shortened such that the measurement at marking at the end of the tube was equal to the value obtained from a 50 pL aliquot that was sampled at the tube opening and diiuted to 1 mL with water (12.5 pg Au/niL) i.e. to a sample where the aggregation was stopped by dilution with water and measured using 785 nm laser excitation and an Océan Optics QE65000 spectrometer set to one second intégration time. The results of the SERS measurements arc plotted in Fig. 5 as diamonds connected by a solid line. The SERS signal intensity from the sample taken at the end of the tube and measured at 12.5 pg Au/mL was 11044 counts per second [± 500].

V.2 First colloid and second colloid contain Au nanoparticles of different sizes (Examples E9 EU)

V,2.a Batch aggregation (Examples E9 and E10)

Example E9

Second colloid (D7, 30mL) was quickly poured into first colloid (Al, 55 mL) and the mixture was stîrred in the mixing réservoir with a magnetic stirrer bar, The ratio between the number of the 5 nanoparticles of the first colloid and the number of nanoparticles of the second colloid is ~6,3:1.

After 30 s of mixing at a pH of approximately 2.9, aggregation was stopped by the addition of a polymer solution, and the resulting aggregates were further silica coated following the method described in US8497131B2 to provide the target SERS tags. ~2 pL aliquots ofthe SERS tags were dropped onto silica wafer pièces and dried. The sample was imagcd on a Hitachi S-4500 field 10 émission SEM and shown in Fig. 3a. The SERS signal was measured using 785 nm laser excitation and a QE65000 spectrometer purchased from Océan Optics Inc. set to one second intégration time. The SERS signal intensity measured at 12.5 pg Au/mL was 18377 counts per second [± 500].

Example E10

Second colloid (D7, 20 mL) was quickly poured into the first colloid (A7, 200 mL) and the mixture was stîrred in the mixing réservoir with a magnetic stirrer bar. The ratio between the number of the nanoparticles of the first colloid and the number of nanoparticles of the second colloid is «2.65:1.

After 30 s of mixing at a pH of approximately 2.9, aggregation was stopped by the addition of a 20 polymer solution, and the resulting aggregates were silica coated following the method described in US8497131B2 to provide the target SERS tags. ~2 pL aliquots ofthe SERS tags were dropped onto silica wafer pièces and dried. The sample was imaged on a Hitachi S-4500 field émission SEM and is shown in Fig. 3b.

V,2.b Flow system aggregation (Example Eli)

A schematic of the continuous flow reactor used in the manufacturing method according to the présent invention is presented in Fig. 1b. A pressurized tank (111b) holds the second colloid, and a second pressurized tank (112b) holds the first colloid. A vessel open to the atmosphère (collecting réservoir, 114b) is used to collect the SERS tags and to stop aggregation. Fluorinated 30 ethylene propylene (1ΈΡ) from Cole Panner® Scientific Experts tubing with 8 mm nominal internai diameter is used for colloid transport. 30 cm of transparent FEP tubing was used to connect the pressurized tank (111b) lo the T-junction (113b). 30 cm of FEP tubing was used to connect the pressurized tank (112b) to the T-junction (113b). 7 m of FEP tubing was used to connect the Tjunction (113b) to the collection vessel (114b). This section of tubing between the T-junction 35 (113b) and the collection vessel (114b) is the section in which aggregation occurred.

The colloids A6 (12 L) and D8 (3 L) described above are placed in tanks 112b and 111b, respectively. The tank (111b) containing second colloid (D8) was pressurized such that the colloid flowrate was 0.66 L/min. The tank (112b) containing the first colloid (A6) was pressurized such 5 that the activated colloid flowrate was 2.65 L/min. After combining at the T-junction, the combined colloid, characterized by a ratio between the number of nanoparticles of the first colloid A6 and the number of nanoparticles of the second colloid D8 is of .2 : 1, has a pH of about 4.8 and a flow rate in the aggregation tubing of 3.31 L/min, giving a linear velocity of 1.1 m/s. SERS measurements were taken tlirough the transparent FEP tubing at 1 m markings down the length of 10 the aggregation tubing with a probe coupled with a fiber optîc cable to an Océan Optics QE65000 spectre me ter with 785 nm excitation. The intégration time was shortened such that the measurement at marking at the end of the tube was equal to value obtained from a 50 pL aliquot that was sampled at the tube openîng and diluted to 1 mL with water (12.5 pg Au/mL) (step e2) and measured using 785 nm laser excitation and an Océan Optics QE65000 spectrometer set to one second intégration time. The results of these measurements are plotted in Fig. 5 as cîrcles connected by a dotted line. The SERS signal intensity from the sample taken at the end of the tube and measured at 12.5 pg Au/mL was 28303 counts per second [± 500].

Claims

1. A method for manufacturing surface enhanced Raman spectroscopy (SERS) tags comprising the steps:

a) providing a first colloid consisting essentially of nanoparticles having a plasmonic surface and substantially same size dispersed in an aqueous solvent, and a stabîlizing agent adsorbed on the suriàce of said nanoparticles, and having a ζ-potential value lower than or equal to -25 mV;

b) providing a second colloid consisting essentially of nanoparticles having a plasmonic surface and substantially same size dispersed in an aqueous solvent, Ratnan active reporter molécules adsorbed on the surface of said nanoparticles, and a stabîlizing agent adsorbed on the surface of said nanoparticles, and having a ζ-potential value lower than or equal to -25 mV;

c) combining the first colloid with the second colloid so that the ratio between the number of nanoparticles of the first colloid and the number of nanoparticles of the second colloid is of between about 25 : 1 to about 1:1, preferably from about 5 : 1 to about 1 : 1 to provide a third colloid;

d) inducing aggregation of the nanoparticles by any of the steps dl) — d3) or a combination thereof:

2. The method according to claim 1, wherein step b) comprises the following steps conducted in the order bl) to b3):

bl) providing a colloid consisting essentially of nanoparticles having a plasmonic surface and substantially same size dispersed in an aqueous solvent, and a stabîlizing agent adsorbed on the surface of said nanoparticles, and having a ζ-potcntial value lower than or equal to -25 mV;

b2) adjusting the pli ofthe colloid at a value higher than the lowest pH value at which the Raman active reporter molécules to be adsorbed on the surface of the nanoparticles carry no net electrical charge while maintaining the ζ-potential vaine lower than or equal to -25 mV, preferably lower than -40 mV; and b3) adding a solution of the Raman active reporter molécules in a solvent to the colloid obtained at step b2) while maintaining the ζ-potential value lower than or equal to -25 mV.

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

4. The method according to any one of claims 1 to 3, wherein the nanoparticles of the second colloid hâve absorbed on their surface a sub-monolayer or monolayer of Raman active reporter molécules.

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

6. The method according to any one of claims 1 to 5, wherein at step c) the ratio between the number of nanoparticles ofthe first colloid and the number of nanoparticles of the second colloid is of between about 4 : 1 and about 3:1.

7. The method according to any one of claims 1 to 6, wherein step dl) further comprises addition of an acid solution to the third colloid obtained at step c) while mixing so that the pH value of the resulting colloid is comprised between about 2.2 and the lowest pH value' at which the Raman active reporter molécules hâve a net electrical charge of between 0 and 0.3.

8. The method according to any one of claims 1 to 6, wherein the pH of the first colloid is adjusted so that the pH of the third colloid obtained at step dl) is comprised between about 2.2 and the lowest pH value at which the Raman active reporter molécules hâve a net electrical charge of between 0 and 0.3.

9, The method according to any one of claims 1 to 8, wherein step e) comprises any of the followings steps el) - e4):

el) adjusting the pH of the colioid obtained at step d) at a value higher than the lowest pH value at which the Raman active reporter molécules to be adsorbed on the surface of the nanoparticles carry no net electrical charge;

e2) diluting the colloid obtained at step d) with water;

e3) addition of a polymer to the colloid obtained at step d);

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

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

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

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

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

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