ES2940580A1 - MICROFLUIDIC CHIP FOR THE IN-SITU SYNTHESIS OF SILVER NANOSTARS WITH PLASMONIC PROPERTIES FOR SERS ANALYSIS (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Microfluidic chip for the in-situ synthesis of silver nanostars with plasmonic properties for sers analysis. The object of the invention is a microfluidic chip for the in-situ synthesis of silver nanostars with plasmonic properties comprising: a first micrometric channel (1) for the entry of a first solution, a second micrometric channel (2) for the entry of a second solution; a first mixing zone (3) for the passive mixing between the first solution and the second solution; a third micrometric channel (4) for the air inlet; a fourth micrometer channel (5) for the entry of a fourth solution; a fifth micrometric channel (6) for the entry of a fifth mixing solution and a second mixing area (7) for the active mixture between the third, fourth and fifth solutions, giving rise to the nanoemulsion of silver nanostars. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

MICROFLUIDIC CHIP FOR THE IN-SITU SYNTHESIS OF NANOSTARS OF

SILVER WITH PLASMONIC PROPERTIES FOR SERS ANALYSIS

TECHNIQUE SECTOR

The present invention belongs to the technical field of microfluidics and, in particular, refers to a new method for the controlled synthesis of silver nanostars with plasmonic properties, as well as their use for SERS analysis of analytes in solution. The microfluidic chip to carry out said method is also an object of the invention.

BACKGROUND OF THE INVENTION

SERS spectroscopy ( Surface-Enhanced Raman Scattering ) is a technique capable of increasing the intensity of the Raman signal of organic compounds, reducing the detection limits by up to ten orders of magnitude, being even capable of detecting individual molecules.

The method consists of performing a Raman analysis on samples diluted or placed in contact with nanometric structures whose plasmonic properties encourage the coordinated vibration of the molecules, producing a resonance effect that amplifies their Raman signal and facilitates their detection. By guaranteeing an extremely low detection limit, the SERS technique finds application in all those fields of study where it is important to identify (or even semi-quantify) organic compounds present in very low concentrations, including the analysis of drugs, explosives, pesticides or food products. , among other examples. Additionally, since numerous active space missions and those in the development phase contemplate the deployment of Raman spectrometers in celestial bodies, the SERS method is considered one of the possible methods to be used for the detection of traces of extraterrestrial life.

Since the year of the discovery of the SERS effect in 1977, the scientific community has proposed a wide variety of plasmonic matrices to be used for the amplification of Raman signals. Among them, conventional SERS analyzes are based on the use of emulsions composed of gold or silver nanospheres. However, knowing that the plasmonic effect is amplified by the roughness of the nanoparticles, more recent developments propose the synthesis of nanoparticles with a highly irregular surface, such as nanostars (Garcia-Leis, A., et al., Journal of Physical Chemistry C 117, 7791-7795, 2013) and nanoflowers (Xie, J., et al., ACS Nano 2, 2473-2480, 2008).

As the pertinent bibliography demonstrates, these complex nanostructures manage to further amplify the Raman signals, allowing the detection limit to be lowered by up to five orders of magnitude with respect to that achieved by conventional SERS emulsions (Garcia-Leis, A., et al., Journal of Physical Chemistry C 117, 7791 7795, 2013; Garcia-Leis, A., et al., Colloids and Surfaces A: Physicochemical and Engineering 535, 49-60, 2017).

Recognizing the potential of its vast applicability, in recent years several national and international patents have been published for different methods of synthesis of SERS nanostars (CN109085150, CN111299570, CN110658176, CN109128152).

Despite greatly lowering the detection limit of organic compounds, one of the major limitations of SERS is the low reproducibility of the analyses. This limitation is mainly due to two factors. On the one hand, since the plasmonic effect is very sensitive to the shape and size of the nanoparticles used, contamination and other undesired variations (for example, small variations in the pH values or in the concentration ratio of the reagents), which can occur during the synthesis process, they can compromise the shape and stability of the colloid, thus affecting the reproducibility of SERS assays (Chem. Sci. 11, 4563-4577, 2020). On the other hand, numerous studies show that the plasmonic amplification of Raman signals is inversely related to the age of the colloid used, since increasing the storage time also increases the probability of oxidation of the nanoparticles and the occurrence of flocculation phenomena. (Journal of Physical Chemistry C., 123, 16495-16507, 2019).

Although different strategies have been tested to limit oxidation and flocculation phenomena, such as the application of stabilizers ( capping agents) or protective layers ( stabilizers ), the lifetime of conventional SERS colloids is typically on the order of a few weeks. or even days (Jiang, Y. et al., Applied Surface Science 450, 451—460, 2018).

If the lifetime guaranteed by conventional SERS colloids is compatible with most laboratory and industrial applications, this durability may not be sufficient in certain fields of study. Taking the field of planetary exploration as an example, no SERS colloid or substrate currently available is capable of guaranteeing optimal performance in the time frames that require space missions to other celestial bodies in the solar system (in the order of months or even years). For this reason, the only possible strategy for a possible application of SERS in planetary missions consists in the in-situ production of nanoemulsions with plasmonic properties.

Known studies have shown that the in-situ production of nanoemulsions with high reproducibility of analysis can be carried out by using microfluidics systems. According to these studies, microfluidics devices allow automating the synthesis process: 1) achieving precise control of aggregation time, 2) reducing mechanical and optical variations of colloids, 3) allowing more efficient mixing between colloid and analyte, 4) minimizing the use of resources and 5) reducing possible contaminations (Journal of Physical Chemistry C 123, 5608-5615, 2019).

The first patent published on the subject describes the use of microfluidic systems for the production of conventional SERS nanospheres, these being obtained by mixing two or more solutions through communicating micrometric channels (WO03038436). However, more recent patents describe more elaborate microfluidics systems (for example, including heating and reaction zones), which allow the automated synthesis of SERS colloids of more complex structure to be achieved. For example, patent application CN110666186 describes a microfluidic chip that makes it possible to synthesize gold nanostars.

In this context, it should be noted that the plasmonic effect of a certain colloid is directly related to the type of source used for excitation of the sample. On the one hand, the Surface Plasmon Resonance (SPR) peak provided by gold colloids optimizes the SERS effect that is achieved by using Raman instruments equipped with red lasers (eg 633 nm). On the other hand, silver colloids optimize the SERS effect when green (532 nm) excitation sources are used (Colloid and Polymer Science 296, 1029-1037, 2018).

Currently, there is no known microfluidic system that makes it possible to synthesize silver nanostars, being the metal whose plasmonic resonance peak best matches the wavelength (532nm) of the excitation source used by Raman instruments that operate in current (SuperCam instrument, aboard the NASA/Perseverance rover) and future space missions (RLS spectrometer, aboard the ESA/Rosalind Franklin rover or RAX instrument, aboard the JAXA/MMX rover). The present invention solves this need.

DESCRIPTION OF THE INVENTION

A first object of the invention is a microfluidic chip for the in-situ synthesis of silver nanostars with plasmonic properties, characterized in that it comprises, on its surface:

• at least one first micrometric channel for the inlet of a first mixing solution, wherein said first micrometric channel is connected to a storage tank for said first mixing solution and has a width of between 50 and 1000 ^m (preferably between 200 and 400 ^m) and a length between 50 and 400 ^m (preferably between 100 and 300 ^m). Preferably, the first micrometric channel will be configured to operate automatically, for which it will be able to have at its input at least one pump or other alternative device to increase the pressure of the mixing solution, such as a syringe. Alternatively, the first micrometer channel will be configured to operate manually;

• at least one second micrometer channel for the input of a second mixing solution, where said second micrometer channel is connected with a storage tank for said second mixing solution and has a width between 50 and 1000 ^m (preferably between 200 and 400 ^m) and a length between 50 and 400 ^m (preferably between 100 and 300 ^m ). Preferably, the second micrometric channel will be configured to operate automatically, for which it will be able to have at its input at least one pump or other alternative device to increase the pressure of the second mixing solution, such as a syringe. . Alternatively, the second micrometer channel will be configured to operate manually;

a first mixing zone connected with the first micrometric channel and with the second micrometric channel, where said first mixing zone is configured to carry out passive mixing between the first solution and the second solution, giving rise to a third mixing solution (corresponding to mixing the first solution and the second solution). Preferably, said first mixing zone will consist of a channel that contains within it a suitable system for the passive mixing of microfluidics, said system being preferably selected from those described in Lee, C.Y. et al., Chemical Engineering Journal 288, 146-160, 2016 (Cross Channel System, Zigzag Channel System, Roll Mix System, Serpentine System, Embedded Barrier System, Herringbone Channel System of fish or system of inclined wells, among others);

at least one third micrometric channel for the inlet of air (or another inert medium in the form of gas or liquid), wherein said third channel is connected to a storage tank for said inert medium and has a width of between 50 and 1000 ^m (preferably between 200 and 400 ^m) and a length between 50 and 400 ^m (preferably between 100 and 300 ^m). Preferably, the third micrometric channel will be configured to operate automatically, for which it will be able to have at its input at least one pump or other alternative device to increase the air pressure (or other inert medium) such as, for example, a syringe. Alternatively, the third micrometer channel will be configured to operate manually, by using, for example, a blister pack;

at least a fourth micrometer channel for the inlet of a fourth mixing solution, wherein said fourth channel is connected to a tank of storage of said fourth mixing solution and has a width between 50 and 1000 ^m (preferably between 200 and 400 ^m) and a length between 50 and 400 ^m (preferably between 100 and 300 ^m). Preferably, the fourth micrometric channel will be configured to operate automatically, for which it may have at its input at least one pump or other alternative device to increase the pressure of the fourth mixing solution, such as a syringe. . Alternatively, the fourth micrometer channel will be configured to operate manually;

• at least a fifth micrometric channel for the inlet of a fifth mixing solution, wherein said fifth micrometric channel is connected to a storage tank for said fifth mixing solution and has a width of between 50 and 500 ^m (preferably between 100 and 200 ^m) and a length between 50 and 200 ^m (preferably between 100 and 200 ^m). Preferably, the fifth micrometric channel will be configured to operate automatically, for which it may have at its input at least one pump or other alternative device to increase the pressure of the fifth mixing solution, such as a syringe. ;

• a second mixing zone connected to the third, fourth and fifth micrometer channel, as well as to the first mixing zone through a connection conduit with said first mixing zone. Said second mixing zone is configured to carry out an active mixing between the third, fourth and fifth solutions, giving rise to the synthesis of a nanoemulsion of silver nanostars. To carry out said mixing, any suitable device for the active mixing of solutions may be used, such as a pressure generating device, ultrasound, dielectrophoretic forces, electrokinetic forces, thermal forces or magnetic forces, among other possibilities. Finally, the second mixing zone also includes an outlet conduit for the nanoemulsion of silver nanostars.

The claimed microfluidic chip allows the conventional nanostar synthesis process to be replicated on a micrometer scale, as described in Garcia-Leis et al., J. Phys. Chem. C 117, 7791-7795, 2013. This is a great advantage, since which makes it possible to optimize resources, which is especially important in the use of the claimed microfluidic chip in space exploration missions, this being one of the main applications of the object of the invention.

Another advantage of the claimed microfluidic chip is that it allows the in situ synthesis of highly reproducible nanoemulsions. Additionally, the silver nanostars obtained from the claimed device and method have a morphology comparable to that of the silver nanostars prepared by the conventional method.

In a particular embodiment of the invention, the developed microfluidic chip may be used both for the in-situ synthesis of silver nanostars with plasmonic properties, and for the SERS analysis of at least one analyte in solution using previously synthesized silver nanostars. In said particular embodiment, the microfluidic chip will comprise, in addition to the elements described above, the following elements:

• at least a sixth micrometric channel for the entrance of the solution to be analyzed (analyte), where said sixth channel is connected to a storage tank for said sixth mixing solution and has a width of between 50 and 1000 ^m (preferably between 100 and 200 ^m) and a length between 50 and 400 ^m (preferably between 100 and 200 ^m). Preferably, the sixth micrometer channel will be configured to operate automatically, for which it will be able to have at its input at least one pump or other alternative device to increase the pressure of the solution to be analyzed, such as a syringe. Alternatively, the sixth micrometer channel will be configured to operate manually;

• a third mixing zone connected to the sixth micrometer channel and to the outlet conduit of the second mixing zone. Said third mixing zone is configured to carry out a passive mixing between the nanoemulsion obtained in the second mixing zone and the solution to be analyzed (analyte). Preferably, said third mixing zone will consist of a channel whose shape and/or structure favors the passive mixing of microfluidics, said system being preferably selected from those described in Lee, CY et al., Chemical Engineering Journal 288, 146 -160, 2016, such as those previously described to carry out the first mix (crossed channel system, zigzag channel system, lamination mixing system, serpentine-shaped system, embedded barrier system, of channels with herringbone design or system of wells inclined, among others);

• at least a seventh micrometer channel connected to the third mixing zone with a width between 50 ^m and 20 mm (preferably between 100 and 300 ^m) and a length between 50 ^m and 10 mm (preferably between 100 and 300 ^ m). Said seventh channel additionally comprises at least one SERS analysis chamber, which implies that at least one of the sides of the chamber is made of a material that is transparent to the excitation laser of the Raman spectrometer used for the SERS analysis of the sample, as well as to the light scattered by it, being able to be selected from at least one polymer, amorphous glass and quartz, among others, preferably being made of low dispersion glass and low diffraction index (for example, borosilicate glass).

In turn, the side of the SERS chamber opposite to the previous one can be metallized (preferably, by means of an aluminum coating), which allows the reflection of the incident laser beam on the sample, thus increasing the energy density that the sample receives. to analyze;

• the seventh channel is connected to at least an eighth output micrometer channel, where said output micrometer channel has a width of between 50 and 1000 ^m (preferably between 200 and 400 ^m) and a length of between 50 and 400 ^m (preferably between 100 and 300 ^m).

Another object of the invention is a method for the controlled synthesis of silver nanostars with plasmonic properties, based on microfluidic technology, where said method is carried out using a microfluidic chip for the in-situ synthesis of silver nanostars with previously plasmonic properties. described and is characterized by the fact that it comprises the following stages: • introducing between 5 and 50 ^l of a first mixing solution that includes hydroxylamine in a concentration of between 1.2^10-2 and 6.0^10-2 M through at least a first micrometer channel with a width of between 50 and 1000 ^m (preferably between 200 and 400 ^m) and a length between 50 and 400 ^m (preferably between 100 and 300 ^m). Preferably, this stage will be carried out automatically, through the use of at least one pump located at the entrance of the first micrometer channel;

• introduce between 5 and 50 ^l of a second mixing solution comprising sodium hydroxide in a concentration between 1.0^10-2 and 5.0^10-2 M through at least a second micrometer channel with a width of between 50 and 1000 ^m (preferably between 200 and 400 ^m) and a length between 50 and 400 ^m (preferably between 100 and 300 ^m). Preferably, this stage will be carried out automatically, through the use of at least one pump located at the entrance of the second micrometer channel;

mixing the first solution and the second mixing solution, in a first mixing zone connected to the first micrometer channel and to the second micrometer channel, giving rise to a third mixing solution. Preferably, the mixing can be favored by using a suitable system for the passive mixing of microfluidics, said system being preferably selected from among those described in Lee, C.Y. et al., Chemical Engineering Journal 288, 146-160, 2016, as described above.

This mixture is then introduced into a second mixing zone connected to the third, fourth, and fifth micrometer channels, as well as to the first mixing zone through a connection conduit with said first mixing zone;

introduce drop by drop into the second mixing zone between 50 and 250 ^l of a fourth mixing solution comprising silver nitrate in a concentration between 1.0^10-3 and 3.6^10-3 M through at least one quarter micrometric channel with a width between 50 and 1000 ^m (preferably between 200 and 400 ^m) and a length between 50 and 400 ^m (preferably between 100 and 300 ^m). Preferably, this stage will be carried out automatically, by using at least one pump located at the inlet of the fourth micrometer channel. Thus, mixing the silver nitrate solution with the hydroxylamine and sodium hydroxide solution will be mixed under stirring.

after between 1 and 30 minutes, preferably between 2 and 10 minutes, introduce into the second mixing zone between 1 and 50 ^l of a fifth mixing solution comprising sodium citrate in a concentration between 1.6 •10-3 and 4.13^ 10-2 M through at least a fifth micrometer channel with a width between 50 and 500 ^m (preferably between 100 and 200 ^m) and a length between 50 and 200 ^m. Preferably, this stage will be carried out automatically, through the use of at least one pump located at the inlet of the fifth micrometer channel;

Preferably, the molarity and the concentration ratio between the solutions described above will be modulated so that the final mixture obtained in the second mixing zone has a pH value between 4.5 and 7 and comprises hydroxylamine in a concentration between 2.0 and 4.0^10-3 M, silver nitrate in a concentration between 0.2 and 1.2^10-3 M, and sodium citrate in a concentration between 0.1 and E10-3 M;

• carry out the active mixing between the third, fourth and fifth mixing solutions in the second mixing zone for a time of at least 5 minutes, preferably between 10 and 20 minutes and more preferably 15 minutes, giving rise to a nanoemulsion. of silver nanostars with plasmonic properties. This mixture may be favored by using a pressure generating device, ultrasound, dielectrophoretic forces, electrokinetic forces, thermal forces or magnetic forces, among other options; • Finally, air (or another inert medium, liquid or gas) is introduced into the second mixing zone through a third micrometric channel with a width of between 50 and 1000 ^m (preferably, between 200 and 400 ^m) and a length between 50 and 400 ^m (preferably between 100 and 300 ^m). Preferably, this stage will be carried out automatically, by using at least one pump located at the inlet of the third micrometer channel. The amount of air (or other inert medium) introduced into the second mixing zone will preferably be equal to or greater than 30% of the volume of said second mixing zone. The air (or other inert medium) introduced into the second mixing zone will favor the exit of the nanoemulsion of silver nanostars obtained in the previous stage through an outlet conduit from said second mixing zone.

Another object of the invention is a method of using the silver nanostars obtained by means of the previously described method for the SERS analysis of at least one analyte in solution. In this way, the previously described method, according to any of its alternatives, can additionally comprise the following stages:

• introduce the solution to be analyzed (analyte) through at least a sixth micrometer channel with a width between 50 and 1000 ^m (preferably between 100 and 200 ^m) and a length between 50 and 400 ^m (preferably between 100 and 200 ^m). Preferably, this stage will be carried out automatically, through the use of at least one pump located at the inlet. of the sixth micrometric channel;

• mix the nanoemulsion obtained in the second mixing zone and the solution to be analyzed (analyte) in a third mixing zone connected to the sixth micrometer channel and to the outlet conduit of the second mixing zone. Preferably, said mixing will be promoted by using a suitable system for passive mixing of microfluidics, such as those described in Lee, C.Y. et al., Chemical Engineering Journal 288, 146-160, 2016;

• leading the mixture of the nanoemulsion and the solution to be analyzed through at least a seventh micrometer channel connected to the third mixing zone, and subjecting the mixture to a SERS analysis by means of a SERS analysis chamber located in said seventh channel;

• once analysed, the sample is led outside through at least an eighth micrometer outlet channel with a width of between 50 and 1000 ^m (preferably between 200 and 400 ^m) and a length of between 50 and 400 ^ m (preferably between 100 and 300 ^m).

The previously described method offers multiple advantages over alternative methods of the state of the art. In particular, it allows a significant saving of reagents, as well as achieving a precise result of the synthesis carried out.

BRIEF DESCRIPTION OF THE DRAWINGS

To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, a set of drawings is attached as an integral part of said description where, for illustrative and non-limiting purposes, the following has been represented :

• Figure 1: Front view of the microfluidic chip of the invention developed for the synthesis of silver nanostars and the SERS analysis of analytes in solution.

• Figure 2: UV-Vis spectrum of the silver nanostars synthesized following the conventional method (a) and using the microfluidic chip represented in Figure 1 (b).

• Figure 3: SEM images of silver nanostars synthesized using the conventional method (a and b) and by means of the microfluidic chip represented in Figure 1 (c and d).

• Figure 4: Conventional Raman spectra of adenine diluted in miNiQ water at different concentrations. The visible bands around 1600 cm-1 and between 3000 and 3600 cm-1 are due to the O-H vibrations of the water.

• Figure 5: SERS spectra of adenine diluted in milliQ water at different concentrations. The analyzes have been carried out using the microfluidic chip in Figure 1. The visible bands around 1600 cm-1 and between 3000 and 3600 cm-1 are due to the O-H vibrations of the water.

DETAILED DESCRIPTION OF THE INVENTION

A particular embodiment of the invention is described below, as shown in Figure 1 that accompanies this description.

Said Figure 1 shows a particular embodiment of the microfluidic chip for the in-situ synthesis of silver nanostars with plasmonic properties, as well as for the SERS analysis of at least one analyte in solution, which comprises:

• at least a first micrometric channel (1) for the entry of a first mixing solution, with a width of 100 ^m and a length of 100 ^m. Said first micrometric channel (1) comprises at its inlet at least one pump with a flow and pressure control system;

• at least a second micrometric channel (2) for the entry of a second mixing solution, with a width of 100 ^m and a length of 100 ^m. Said second micrometric channel (2) comprises at its inlet at least one pump with a flow and pressure control system;

• a first mixing zone (3) connected to the first micrometric channel (1) and to the second micrometric channel (2) consisting of a channel with a width of 200 ^m and a length of 100 ^m containing inside a staggered herringbone structure;

• at least a third micrometric channel for the air inlet (4), with a width of 200 ^m and a length of 100 ^m;

• at least a fourth micrometric channel (5) for the input of a fourth solution of mixture, with a width of 200 ^m and a length of 100 ^m. Said fourth micrometric channel (5) comprises at its entrance at least one pump with a flow and pressure control system;

• at least a fifth micrometric channel (6) for the inlet of a fifth mixing solution, with a width of between 50 ^m and a length of 50 ^m. Said fifth micrometric channel (6) comprises at its inlet at least one pump with a flow and pressure control system;

• a second mixing zone (7) connected to the third, fourth and fifth micrometer channels (4, 5, 6), as well as to the first mixing zone through a connection pipe with said first mixing zone (3 ). Said second mixing zone (7) is made up of a mixer driven by a stirring bar with a capacity of 250 ^l;

• at least a sixth micrometric channel (8) for the entrance of the solution to be analyzed (analyte), with a width of 200 ^m and a length of 100 ^m. Said sixth micrometric channel (8) comprises at its inlet at least one pump with a flow and pressure control system;

• a third mixing zone (9) connected to the sixth micrometric channel (8) and to the outlet conduit of the second mixing zone (7). Said third mixing zone (9) consists of a channel with a width of 400 ^m and a length of 100 ^m that contains in its interior a structure of herringbone fish (in English, staggered herringbone structure );

• at least a seventh micrometer channel connected to the third mixing zone (9), where said seventh channel comprises at least one SERS analysis chamber (10). Said seventh channel has a width of 200 and a length of 300 ^m. Both the upper face and the lower face of the SERS analysis chamber (10) are made of quartz;

• Finally, the SERS analysis chamber (10) is connected to at least an eighth output micrometer channel (11).

Next, the method of controlled synthesis of silver nanostars with plasmonic properties is described, as well as the SERS analysis of at least one analyte in solution carried out using the previously described micrometer chip. In particular, said method is characterized by the fact that it comprises the following steps:

• introduce 12 ^l of a first mixing solution comprising hydroxylamine in a 6^10-2 M concentration through the first micrometric channel (1) by means of the pump with flow and pressure control located at the inlet of said first channel;

introduce 12 ^l of a second mixing solution comprising sodium hydroxide in a 5^10-2 M concentration through the second micrometric channel (2) by means of the pump with flow and pressure control located at the inlet of said second channel ;

passively mixing the first solution and the second mixing solution, in a 1:1 ratio, in the first mixing zone (3), giving rise to a mixture that is sent to the second mixing zone (7). The flow and pressure at the inlet of the first micrometer channel (1) and the second micrometer channel (2) are controlled to introduce 20 ^l of the third solution (obtained from the mixture of the first solution and the second solution) to the second mixing zone (7); introduce drop by drop into the second mixing zone (7) 216 ^l of a fourth solution comprising silver nitrate in a concentration E10-3 M through the fourth micrometric channel (5), where the flow and pressure of said fourth solution are controlled by the pump located at the entrance of said channel;

After five minutes, introduce into the second mixing zone (7) 6 ^l of the fifth mixing solution comprising sodium citrate in a concentration of 4.13^10-2 M through the fifth micrometric channel (6), where the flow and pressure of said fifth solution are controlled by means of the pump located at the entrance of said channel;

carry out the active mixing between the third, fourth and fifth mixing solutions in the second mixing zone (7), giving rise to a nanoemulsion of silver nanostars with plasmonic properties. This active mix is favored by using a stirring bar contained inside the second mixing zone (7);

After fifteen minutes, introduce air into the second mixing zone (7) through the third channel (4), favoring the expulsion of the nanoemulsion of silver nanostars from the second mixing zone (7) towards a third mixing zone ( 9);

introduce the solution to be analyzed (analyte) through the sixth micrometric channel (8) by means of the pump located at the entrance of said channel;

mix the nanoemulsion obtained in the second mixing zone (7) and the solution to be analyzed (analyte) in the third mixing zone (9) and driving the mixture obtained through the seventh channel that includes the SERS analysis chamber (10);

• carry out the SERS analysis by applying an excitation source from the Raman spectrometer through the transparent material window of the SERS analysis chamber (10) and, once the desired SERS spectra are obtained, lead the mixture to the outside through the eighth micrometer channel (11).

To demonstrate the technical effect achieved by the claimed micrometer chip and method, a series of tests were carried out to evaluate the morphological and plasmonic characteristics of the synthesized silver nanostars. For this, the nanoemulsion generated in the second mixing zone (7) was analyzed using a UV-vis spectrometer and a scanning electron microscope (SEM). The results obtained were compared with the silver nanoparticles generated following the conventional method described in Garcia-Leis et al., J. Phys. Chem. C 117, 7791-7795, 2013.

Figure 2 shows a comparison between the absorbance spectrum of the nanoemulsion prepared with the conventional method (a) with that synthesized using the previously described microfluidic chip, object of the present invention (b). Both spectra show the maximum absorbance point at 376 nm and a minimum around 346 nm. The profile of the two spectra fits perfectly with the UV-Vis results presented in Garcia-Leis et al., J. Phys. Chem. C 117, 7791 7795, 2013, confirming the synthesis of high-quality silver nanostars.

SEM analyzes were carried out in order to determine the morphology of the synthesized silver nanostars. As can be seen in Figure 3, the nanostars described in Garcia-Leis et al., J. Phys. Chem. C 117, 7791-7795, 2013 (Figures 3a and 3b) are between 300 and 400 nm in size. As detailed in the work of Garcia-Leis et al., Colloids Surfaces A Physicochem. Eng. Asp. 535, 49 60, 2017, the number and complexity of the ramifications of each nanostar depends, among other factors, on the degree of maturity of the sample. As can be seen in Figures 3c and 3d, the size and shape of the nanostars synthesized using the claimed microfluidic chip coincide with those obtained using the conventional method.

Once the effectiveness of the method for the synthesis of silver nanostars with the desired morphological properties was confirmed, a series of additional studies were carried out to determine their plasmonic effect. For this, the detection limit reached by means of a conventional Raman analysis was compared with that provided by the nanoemulsion synthesized by means of the claimed microfluidic chip. In particular, adenine was used as a reference biomarker. Additionally, the compound was dissolved in milliQ water in different concentration ranges (from 10 -1 to 10 -9 M). In the first experiment, the solutions were introduced into the microfluidic chip through the sixth micrometer channel (8) and were directed directly towards the SERS analysis chamber (10), where they were analyzed using a spectrometer equipped with a 532 nm laser. this being the wavelength used by most of the Raman instruments developed for planetary exploration missions. As can be seen in Figure 4, conventional Raman analyzes allowed the detection of the biomarker up to a concentration of E10-2 M (the reference peak of adenine is detected at 733 nm).

In an additional experiment, the microfluidic chip was used to synthesize the silver nanostar emulsion and mix it with the adenine solution in the third mixing zone (9), before reaching the SERS analysis chamber (10). SERS analyzes carried out using the same Raman spectrometer described above allowed the biomarker to be detected up to a concentration of E10-9 M.

The comparison between the results shown in Figures 4 and 5 show that, using the claimed microfluidic chip, it is possible to produce nanostars whose plasmonic properties allow SERS analysis to be carried out with a detection limit 6 orders of magnitude lower than those conventional Raman analysis.

Claims (9)

1. Microfluidic chip for the in-situ synthesis of silver nanostars with plasmonic properties, characterized in that it comprises, on its surface: (a) at least a first micrometric channel (1) for the entry of a first mixing solution, where said first micrometric channel is connected to a storage tank for said first mixing solution and has a width of between 50 and 1000 ^m and a length of between 50 and 400 ^m; (b) at least one second micrometric channel (2) for the inlet of a second mixing solution, wherein said second micrometric channel is connected to a storage tank for said second mixing solution and has a width between 50 and 1000 ^m and a length between 50 and 400 ^m; (c) a first mixing zone (3) connected to the first micrometric channel (1) and to the second micrometric channel (2), where said first mixing zone (3) is configured to carry out passive mixing between the first solution and the second mixing solution, giving rise to a third mixing solution; (d) at least one third micrometric channel (4) for the inlet of air or another inert medium in the form of gas or liquid, wherein said third channel is connected to a storage tank for said inert medium and has a width of between 50 and 1000 ^m and a length between 50 and 400 ^m; (e) at least a fourth micrometric channel (5) for the inlet of a fourth mixing solution, wherein said fourth channel is connected to a storage tank for said fourth mixing solution and has a width of between 50 and 1000 ^m and a length between 50 and 400 ^m; (f) at least a fifth micrometric channel (6) for the inlet of a fifth mixing solution, wherein said fifth micrometric channel is connected to a storage tank for said fifth mixing solution and has a width of between 50 and 500^ m and a length between 50 and 200 ^m; (g) a second mixing zone (7) connected with the third, fourth and fifth micrometric channel (4, 5, 6), as well as with the first mixing zone (3) through a connection conduit with said first mixing zone (3), where said second mixing zone (7) is configured to carry out an active mixing between the third, fourth and fifth mixing solutions, giving rise to a nanoemulsion of silver nanostars. 2. Microfluidic chip according to claim 1, wherein the first micrometer channel (1) or the second micrometer channel (2) or the third micrometer channel (4) or the fourth micrometer channel (5) or the fifth micrometer channel (6 ) or any of their combinations comprises at least one pump configured to operate automatically. 3. Microfluidic chip according to claim 1 or 2, wherein the first mixing zone (3) consists of a channel that contains within it a suitable system for the passive mixing of microfluidics selected from a group consisting of a cross flume system, zigzag flume system, roll mix system, serpentine system, embedded barrier system, herringbone flume system, and inclined well system. 4. Microfluidic chip according to any one of the preceding claims, wherein the second mixing zone (7) comprises a suitable device for the active mixing of solutions selected from a group consisting of a pressure-generating device, ultrasound, dielectrophoretic forces , electrokinetic forces, thermal forces and magnetic forces. 5. Microfluidic chip according to any one of the preceding claims, wherein said microfluidic chip is additionally suitable for carrying out a SERS analysis of at least one analyte in solution, further comprising: (h) at least a sixth micrometric channel (8) for the entrance of the analyte in solution, wherein said sixth micrometric channel (8) is connected to a storage tank for the analyte in solution and has a width of between 50 and 1000 ^m and a length between 50 and 400 ^m; (i) a third mixing zone (9) connected with the sixth micrometric channel (8) and with the outlet conduit of the second mixing zone (7), where said third mixing zone (9) is configured to carry out carry out a passive mixing between the nanoemulsion obtained in the second mixing zone (7) and the analyte in solution; (j) at least a seventh micrometer channel connected to the third mixing zone with a width between 50 ^m and 20 mm and a length between 50 ^my 10mm, where said seventh channel additionally comprises at least one SERS analysis chamber (10), where at least one of its sides is made of a material that is transparent to incident light on the sample to be analyzed, as well as to light scattered by the same; and (k) at least one eighth micrometer outlet channel (11), connected to the seventh micrometer channel, wherein said micrometer outlet channel (11) has a width of between 50 and 1000 ^m and a length of between 50 and 400 ^m . 6. Microfluidic chip according to claim 5, wherein the seventh micrometer channel and/or the eighth micrometer output channel (11) comprises at least one pump configured to operate automatically. 7. Microfluidic chip according to any one of the preceding claims, wherein the third mixing zone (9) consists of a channel that contains within it a suitable system for the passive mixing of microfluidics selected from a group consisting of a system cross channel system, zigzag channel system, roll mix system, serpentine system, embedded barrier system, herringbone channel system, and inclined well system. 8. Method of controlled synthesis of silver nanostars with plasmonic properties, based on microfluidic technology, where said method is carried out by means of a microfluidic chip according to any one of claims 1 to 4 and where said method is characterized in that comprises the following stages: (a) introducing between 5 and 50 ^l of a first mixing solution comprising hydroxylamine in a concentration between 1.2^10-2 and 6.0^10-2 M through the first micrometer channel (1); (b) introducing between 5 and 50 ^l of a second mixing solution comprising sodium hydroxide in a concentration between 1.0^10-2 and 5.0^10-2 M through the second micrometer channel (2); (c) mixing the first solution and the second mixing solution in a first mixing zone (3), giving rise to a third mixing solution, which is then introduced into the second mixing zone (7); (d) introduce drop by drop into the second mixing zone (7) between 50 and 200 ^l of a fourth mixing solution comprising silver nitrate in a concentration between 1.0^10-3 and 3.6^10-3 M through the fourth micrometric channel (5), the mixing being carried out under stirring of the silver nitrate solution with the mixture the solution of hydroxylamine and sodium hydroxide; (e) after between 1 and 30 minutes, introduce into the second mixing zone (7) between 1 and 50 ^l of a fifth mixing solution comprising sodium citrate in a concentration between 1.6 •10-3 and 4.13^10 -2 M through the fifth micrometer channel (6); (f) carrying out the active mixing between the third, fourth and fifth mixing solutions in the second mixing zone (7) for a time of at least 5 minutes, giving rise to a nanoemulsion of silver nanostars with plasmonic properties; (g) finally, introducing into the second mixing zone (7) air or another inert medium, liquid or gas, through the third micrometric channel (4), favoring the exit of the nanoemulsion of silver nanostars from the second mixing zone. mix (7). 9. Method of using the silver nanostars obtained by the method according to claim 8 for the SERS analysis of at least one analyte in solution, wherein said method is characterized in that it additionally comprises the following steps: (h) introducing the analyte in solution through the sixth micrometric channel (8); (i) mixing the nanoemulsion obtained in the second mixing zone (7) and the analyte in solution in the third mixing zone (9); (j) conducting the mixture of the nanoemulsion of silver nanostars and the solution to be analyzed through the seventh micrometric channel and subjecting the mixture to a SERS analysis using the SERS analysis chamber (10) located in said seventh channel; (k) once analyzed, lead the mixture to the outside through the eighth micrometer outlet channel (11).