WO2021094891A1

WO2021094891A1 - Process for the production of ultra-small pt nanocrystals with high percentage of {111} surface domains

Info

Publication number: WO2021094891A1
Application number: PCT/IB2020/060515
Authority: WO
Inventors: Valentina MASTRONARDI; Mauro MOGLIANETTI; Pier Paolo Pompa
Original assignee: Fondazione Istituto Italiano Di Tecnologia; Universita' Degli Studi Di Genova
Priority date: 2019-11-11
Filing date: 2020-11-09
Publication date: 2021-05-20
Also published as: EP4058223A1; IT201900020697A1

Abstract

The present invention relates to a method for producing Pt nanoparticles with octahedral and/or truncated octahedral shape, having an average size below 4 nm. The method of the invention comprises the steps of: (a) mixing an aqueous solution of a Pt precursor with pre-heated water; 5 (b) adding, after a first waiting time, an aqueous solution of a first reducing agent and, after a second waiting time an aqueous solution of a second reducing agent to obtain a reaction mixture; (c) putting the reaction mixture under a determined pressure, while maintaining the reaction mixture under a reduced oxygen atmosphere and 10 wherein the concentration of dissolved oxygen in the reaction mixture is less than the concentration of oxygen in an oxygen saturated one. The method of the present invention is a seed growth-free method which does not include a step of growing nanoparticle from nanoparticles seeds. The present invention also relates to Pt nanoparticles having an average 15 size below 4 nm, octahedral and/or truncated octahedral shape and a percentage of {111} surface domains comprised between 15 and 35%, preferably between 18 and 24%.

Description

“Process for the production of ultra-small Pt nanocrystals with high percentage of {111 } surface domains”

DESCRIPTION

TECHNICAL FIELD

The present invention relates to a method for producing Pt nanoparticles having controlled shape and size, which does not involve the use of Pt seed growth nanoparticles. The present invention also relates to Pt nanoparticles obtained with the method and their use in diagnostics.

BACKGROUND ART

The possibility to control the shape of nanoparticles, in particular metal nanoparticles, has attracted great attention as the shape dictates the surface arrangement of the atoms and, hence, strongly modulates and enhances specific properties: from improved selectivity and activity in catalytic processes to tunable interaction with light and living matter.

Even though there are several methods available in the literature to obtain specific surface arrangement on the surface of metal nanoparticles by controlling the shape of the nanomaterial, there are few reports on the synthesis of noble metal shaped nanoparticles without the use, as shape directing agents, of polymers, surfactants, organic solvents and other organic molecules difficult to remove after synthesis. For catalytic nanomaterials this is a pivotal aspect. For example, it has been clearly established that, for catalytic Pt nanocrystals, the shape plays a fundamental role as it establishes the surface structure of the atoms, i.e. the specific arrangement of the atoms at the surface, which is the key point determining and controlling the electrocatalytic properties of the material. Many synthetic strategies have been reported in the art to obtain shaped Pt nanocrystals with preferential surface structure. The most widely employed strategy is the stabilization of a facet by using additives, i.e. molecules that selectively stabilize specific surface domains, influence the growth along specific directions, tailoring the final shape of nanoparticles. There is a long list of polymers, surfactants, inorganic salts and small organic molecules that have been used to promote nanocrystals shape formation. However, the coating remaining on the nanocrystal surface affects and often deteriorates the catalytic properties of the material. In particular, it has been clearly established that shape-directing agents such as polyvinylpyrrolidone (PVP), tetradecyl trimethyl ammonium bromide, and oleylamine, poison the catalytic properties of the material by altering its surface properties. For this reason, several purification steps have been proposed to effectively remove the organic coating on the surface of the nanoparticles thus obtained. Nevertheless, most of these treatments are time-consuming, costly, do not guarantee complete removal and, more importantly, may interfere with the surface structure and, hence, with the catalytic properties of the nanocrystals. Moreover, a major challenge in the synthesis of surface-tailored catalytic nanomaterials is that, as the particle size decreases, the proportion of specific surface domains, like {111} and {100}, tends to decrease dramatically, while low coordination sites such as edges, steps, corners and kinks become predominant in the surface. Consequently, the possibility of designing Pt nanoparticles with a controlled shape (i.e. octahedral and/or truncated octahedral shape with a consequent high percentage of {111}) and size (i.e. few nanometers), still represent a challenge in the sector.

Recently, several attempts have been made to synthesize small or ultra small Pt nanoparticles having different shapes.

Document US20180221961 A1 discloses a method for producing a metal nano-alloy with a controlled shape and size, comprising contacting a growth solution, made of a reducible metal precursor and one or more surfactants, with a reducing fluid in a continuous flow reactor to form a mixed solution through the continuous flow reactor and eventually yield the metal nano-alloy. According to said document, a selection of the appropriate surfactant - such as a small-molecules ligand or a polymer surfactant having functional groups capable of coordinating with the metal nano-alloy - is made in order to control the nano-alloy shape and size. Document US8652232B2 discloses a seed-growth based process for preparing cubic metallic nanoparticles comprising: a) preparing an aqueous solution containing a source of a metal from group VIII, a reducing agent R1 and a stabilizer (i.e. a surfactant or a complexing agent); b) preparing an aqueous solution containing a source of a metal from group VIII and a stabilizer (i.e. a surfactant or a complexing agent) at a temperature higher than 70° C and less than or equal to 80°C; c) mixing at least one portion of the aqueous solution of seeds obtained in step a) with the aqueous solution obtained in step b) to obtain the growth, in the presence of a reducing agent R2, of metallic nanoparticles having a cubic shape.

However, the above-mentioned processes not only have the disadvantage of involving multiple-steps synthesis protocols but also employ surfactants as shape promoting agents, which could be detrimental to the final properties of the nanoparticles. An easier and more “green” approach for the synthesis of noble metal nanoparticles, in particular Pt nanoparticles, having an octahedral shape without the use of surfactants and organic solvents as shape directing agents has been proposed by the Applicant in a previous patent application W02017103807A1 , wherein a seed-growth based method for the synthesis of metal nanoparticles of controlled shapes (cubes, cuboids, octahedrons) and size in an aqueous environment without the use of shape directing agent is disclosed. Said method comprises providing a solution comprising the metal seed growth nanoparticles, a metal salt comprising the same metal as the metal seed growth nanoparticles, and a reducing agent and heating the solution under pressure and in a reduced oxygen atmosphere (e.g. by employing a closed vessel). The method for producing said metal seed growth nanoparticles is described, for example, by Moglianetti et al. in “Citrate-coated, size tunable octahedral platinum nanocrystals: a novel route for advanced electrocatalysf , ACS Applied Materials and Interfaces, 2018. According to this paper, Pt seed can be synthesized by adding an aqueous solution of a Pt salt to water at boiling temperature and then adding a solution of sodium citrate and citric acid immediately followed by a quick addition of a solution of NaBhU in an open reaction vessel. Alternative, after the addition of a solution of sodium citrate and citric acid, the solution is immersed in ice to slow down the seed growth and, after the addition of a solution of NaBhU, the reaction is conducted in a closed reaction vessel at 100 °C. Despite the advances in the synthesis of nanoparticles having controlled shape and size, there still exists the need in the art for simple, eco-friendly methods of obtaining preferentially shaped nanoparticles that are preferably free of contaminants (e.g. organic solvents and/or shape directing agents such as polymers and/or surfactants which strongly bind to the surface and are therefore difficult to remove after synthesis ) and that can be obtained with a single-step synthesis which does not include a seed-growth step as instead described in the prior art.

The present invention solves the criticalities of the known art by providing a single-step seed growth-free method for producing ultra-small Pt nanoparticles having octahedral and/or truncated octahedral shape (therefore, with high percentage of {111} surface domains), in an aqueous environment, preferably without the use of organic solvents and/or shape directing agents that are difficult to remove after synthesis, such as polymers and/or surfactants.

SUMMARY OF THE INVENTION

The present invention relates to a method for producing Pt nanoparticles with octahedral and/or truncated octahedral shape, having an average size - measured with transmission electron microscopy - below 4 nm, preferably below 3.8 nm, more preferably below 3.3 nm.

The method of the invention comprises the steps of:

(a) mixing an aqueous solution of a Pt precursor with water, said water being pre-heated at a temperature between 60 °C and 100 °C, preferably between 75 and 95 °C;

(b) after a first waiting time, adding an aqueous solution of a first reducing agent and, after a second waiting time, an aqueous solution of a second reducing agent to obtain a reaction mixture;

(c) putting the reaction mixture under a pressure of between 0.5 and 3 atm, while maintaining the reaction mixture under a reduced oxygen atmosphere, wherein the concentration of dissolved oxygen in the reaction mixture is less than the concentration of oxygen in an oxygen saturated reaction mixture.

Preferably, the method of the present invention does not comprise employing an organic solvent and/or a shape directing agent that is difficult to remove after synthesis, such as a polymer and/or a surfactant. The method of the present invention is a seed growth-free method which does not include a step of growing nanoparticle seeds.

The present invention also relates to Pt nanoparticles having an average size below 4 nm, octahedral and/or truncated octahedral shape, and a percentage of {111} surface domains comprised between 15 and 35%, preferably between 18 and 24 %. The present invention further relates to said Pt nanoparticles for use in diagnostics, preferably for use in an immunoassay.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a bright-field transmission electron microscopy (BF-TEM) image of the Pt nanoparticles obtained according to Example 1.

Figure 2 shows the size distribution of the Pt nanoparticles obtained according to Example 1. Figure 3 shows high-angle annular dark-field scanning transmission electron microscopy (HAADF STEM) images of the Pt nanoparticles obtained according to Example 1.

Figure 4 shows high-resolution transmission electron microscopy (H R- TEM) images of the Pt nanoparticles obtained according to Example 1. Figure 5 shows bright-field transmission electron microscopy (BF-TEM) images of the nanoparticles obtained according to the teachings of the prior art document Moglianetti et al. “Citrate-coated, size tunable octahedral platinum nanocrystals: a novel route for advanced electrocatalysf , ACS Applied Materials and interfaces, 2018, as described in Example 2.

Figure 6 shows bright-field transmission electron microscopy (BF-TEM) images of the nanoparticles obtained according to the teachings of the prior art document Moglianetti et al. “Citrate-coated, size tunable octahedral platinum nanocrystals: a novel route for advanced electrocatalysf, ACS Applied Materials and interfaces, 2018, as described in Example 3.

Figure 7 shows a bright-field transmission electron microscopy (BF-TEM) image of the Pt nanoparticles obtained according to Example 1 deposited on a conductive amorphous carbon substrate as described in Example 5. Figure 8 shows positive sweep scan for Bi desorption on the octahedral Pt nanoparticles obtained according to Example 1. Test solution: 0.5 M FI2SO4. Sweep rate: 50 mVs ¹.

Figure 9 shows the UV-vis absorption curves at 652 nm of the TMB-FI2O2 reaction system catalyzed by Pt ultra-small octahedral nanocrystals (diamond symbol) and Pt nanoparticles synthesized as described in Example 3 (triangle symbol), both kept at a concentration of 0.005 ppm. Figure 10 shows the UV-vis absorption curves at 652 nm of the TMB-FI2O2 reaction system catalyzed by Pt ultra-small octahedral nanocrystals bound to CD195 antibodies (diamond symbol) and spherical Pt nanoparticles synthesized as described in Example 3 bound to CD195 antibodies (triangle symbol).

DETAILED DESCRIPTION OF THE INVENTION For the purposes of the present invention, the term “nanoparticle” can be also intended as a synonym of “nanocrystal”.

For the purposes of the present invention, the term “seed growth-free method” or “seed growth-free synthesis” refers to a process which does not include a seed-growth step, namely a step comprising the use of “metal seed growth nanoparticles”. As described herein, the term “metal seed growth nanoparticles” means a single crystal nanoparticle or a multiple twinned crystal having a certain crystal system from which it is possible to form a larger nanoparticle.

For the purposes of the present invention, “Pt nanoparticle” refers to a nanoparticle of metallic Pt, which means that Pt is present exclusively in the “0” oxidation state (Pt°).

As described herein, the term “shape-directing agent that is difficult to remove after synthesis” means a compound, such as a polymer and/or a surfactant, that is able to influence the growth of a metal nanoparticle by firmly and strongly bonding to one or more of its surfaces.

As described herein, the expression “average size”, when referred to the Pt nanoparticles with octahedral and/or truncated octahedral shape of the present invention, means “average lateral size” (as clearly visible, for example, in Figure 4) and it is determined as described in Example 4, more specifically, by manual imposing a threshold on the HAADF-STEM images followed by automatic measurement of the Feret's diameter using the ImageJ software.

In the context of the present invention, the term “Feret’s diameter” is substituted with the more generic “average size” or “average lateral size” to indicate the dimension of the shaped nanoparticles with the same meaning used by Xia et al. “Shape-Controlled Synthesis of Colloidal Metal Nanocrystals: Thermodynamic versus Kinetic Products” J. Am. Chem. Soc. 2015, 137, 7947-7966.

The present invention refers to a method for the synthesis of Pt nanoparticles with controlled shape and size.

In particular, the present invention refers to a seed growth-free method for producing Pt nanoparticles with octahedral and/or truncated octahedral shape, having an average size - measured with transmission electron microscopy - below 4 nm, preferably below 3.8 nm, more preferably below 3.3 nm, said method comprising the steps of:

(a) mixing an aqueous solution of a Pt precursor with water, said water being pre-heated at a temperature between 60 °C and 100 °C, preferably between 75 °C and 95 °C;

(b) adding, after a first waiting time, an aqueous solution of a first reducing agent and, after a second waiting time an aqueous solution of a second reducing agent to obtain a reaction mixture;

Preferably the aqueous solution of a Pt precursor comprises said Pt precursor in order to reach a concentration in the final solution in the range between 0.01 and 30 mM, preferably between 0.03 and 10 mM, even more preferably between 0.1 and 0.9 mM.

Preferably said Pt precursor is selected from the group consisting of a salt, a complex salt or an acid of Pt, soluble in water.

In a particularly preferred embodiment of the present invention, said Pt precursor is preferably selected from a salt or a complex salt of platinum, more preferably selected from H2PtCl6, Na2PtCl6, K2PtCl6, HQOI2N2RΪ, PtCI₂, PtBr₂, Li₂PtCU, H Pt(OH)₆, Pt(N0₃)2, PtS0₄, Pt(HS0₄)₂, Pt(CI0₄)₂, K2PtCI₄, (NH RΐOIb and combination thereof and hydrates thereof or a mixture of salts and/or hydrates thereof. Even more preferably, the Pt precursor is hexachloroplatinic acid (H^RΐOIb).

Preferably the aqueous solution of a Pt precursor of step (a) is added in an amount comprised between 0.025 and 5 ml, preferably between 0.5 and 5 ml, more preferably between 0.025 and 0.1 ml, even more preferably between 0.04 and 0.08 ml.

Preferably the water of step (a) is selected from the group consisting of: distilled water, deionized water, demineralized water, Milli-Q water. According to a particularly preferred embodiment of the present invention, the water of step (a) is Milli-Q water.

Preferably said water of step (a) is in an amount comprised between 20 ml and 10 L, preferably between 50 ml and 5 L, even more preferably between 70 ml and 100 ml.

Preferably said water of step (a), which is pre-heated at a temperature between 60 and 100 °C, preferably between 75 and 95 °C, is maintained at said temperature during the whole reaction.

This means that the reaction mixture obtained after step b) and employed in step c) is also at a temperature between 60 and 100 °C, more preferably between 75 and 95 °C, said temperature being maintained constant (i.e. without abrupt and/or significative temperature oscillation or changing) from step a) to step c) of the process according to the present invention.

Preferably, in step (b), the aqueous solution of a first reducing agent is added, after a first waiting time, in an amount comprised between 0.05 ml and 50 ml, preferably between 1 and 20 ml, even more preferably between 2 and 5 ml.

Preferably, said first waiting time is comprised between 0.5 and 30 minutes, preferably between 0.8 and 10 minutes.

Preferably, in step (b), the aqueous solution of the first reducing agent comprises said first reducing agent in a concentration comprised between 20 mM and 200 mM, preferably between 30 mM and 50 mM. Preferably, said first reducing agent is an organic molecule having less than 8 carboxylic acids and/or carboxylate groups. Preferably, said first reducing agent also has less than 6 hydroxyl groups.

More preferably, said first reducing agent has no more than 10 functional groups. Preferably, the first reducing agent, does not contain a functional group that strongly binds to the surface of a noble metal such as Pt, said disfavored functional group include one or more functional groups selected from the group consisting of: amine, thiol, phosphine, amide group and combination thereof.

According to a preferred embodiment of the invention, the first reducing agent does not contain an amino functional group.

According to a particularly preferred embodiment of the invention, the first reducing agent is a “small organic molecule”, wherein the term “small organic molecule” refers, for the purposes of the present invention, to an organic molecule having no more than 10 functional groups, less than 8 carboxylic acids and/or carboxylate groups, less than 6 hydroxyl groups and no amino functional groups.

Preferably, the first reducing agent is selected from the group consisting of sodium citrate, citric acid, formic acid, pyruvic acid, oxalic acid, maleic acid, organic molecules which are formed during the oxidation process of sodium citrate such as acetone and formaldehyde, and combination thereof.

According to an embodiment of the present invention, the first reducing agent can be selected also from a variety of organic acids with properties and mechanism of electron transfer comparable to those of sodium citrate, such as pyruvic acid, oxalic acid and formic acid.

According to a particularly preferred embodiment of the present invention, the first reducing agent is a combination of sodium citrate and citric acid. According to said embodiment, the aqueous solution of the first reducing agent is therefore an aqueous solution comprising sodium citrate and citric acid. Preferably, said sodium citrate is present in the final reaction solution volume in a concentration comprised between 0.01 mM and 80 mM, more preferably between 0.08 mM and 20 mM, even more preferably between 0.1 and 0.9 mM. Preferably said citric acid is present in the final reaction solution volume in a concentration comprised between 0.008 mM and 80 mM, more preferably between 0.01 mM and 5 mM, even more preferably between 0.02 and 0.2 mM.

Preferably, in step (b), the aqueous solution of a second reducing agent is added, after a second waiting time, in an amount comprised between 0.01 ml and 100 ml, preferably between 0.5 and 5 ml.

Preferably, the aqueous solution of a second reducing agent is added immediately after the addition of the aqueous solution of a first reducing agent, said second waiting time being therefore preferably comprised between 0.1 and 20 seconds, preferably between 2 and 5 seconds. Preferably, in step (b), the aqueous solution of the second reducing agent comprises said second reducing agent in a concentration comprised between 0.5 mM and 100 mM, preferably between 5 mM and 50 mM. Preferably, the second reducing agent is selected from the group consisting of: sodium borohydride, lithium borohydride, lithium aluminum borohydride, lithium triethylborohydride, diisobutylaluminium hydride and combinations thereof.

According to an embodiment of the present invention, the second reducing agent can be selected also from a variety of hydrides with properties and mechanism of electron transfer comparable to those of sodium borohydride, such as lithium borohydride and lithium aluminum borohydride.

According to a particularly preferred embodiment of the present invention, the second reducing agent is sodium borohydride.

According to said embodiment, the aqueous solution of the second reducing agent is therefore an aqueous solution comprising sodium borohydride. Preferably the aqueous solution of sodium borohydride is added in an amount comprised between 0.05 ml and 15 ml, preferably between 1 and 3 ml at a concentration comprised between 0.5 mM and 100 mM, preferably between 5 and 50 mM.

Preferably, said sodium borohydride is present in final reaction solution volume in a concentration comprised between 0.07 mM and 130 mM, more preferably between 0.1 mM and 1 mM.

According to a particularly preferred embodiment of the present invention, the first reducing agent is a combination of sodium citrate and citric acid and the second reducing agent is sodium borohydride.

Preferably, the first reducing agent is present in the reaction mixture obtained after step (b) in a final concentration between 0.01 mM and 90 mM, preferably between 0.08 mM and 30 mM.

Preferably, the second reducing agent between 0.07 mM and 130 mM, more preferably between 0.1 mM and 1 mM.

Applicant has found that the use of a combination of the above-mentioned first and second reducing agent, preferably when sodium citrate and citric acid are used as the first reducing agent and when sodium borohydride is used as the second reducing agent, is particularly advantageous to produce the nanoparticles of the invention with the desired shape and, in particular, with the desired size.

Without wishing to be bound to a specific theory, it can be envisaged that, as the first reducing agent according to the present invention, for example citrate, has carboxylic and hydroxyl groups, they weakly bind the surface of the Pt nanoparticles. This is a major advantage as they effectively stabilize the nanoparticles in solution against aggregation but, at the same time, they can be easily removed with simple washing with water. In this way it is possible to achieve ultra-small shaped nanoparticles with average size below 4 nm, preferably below 3.8 nm, more preferably below 3.3 nm, and with highly clean surface without losing in stability and without major aggregation processes, which is a crucial aspect for catalysis. Preferably, the pressure of step (c) is comprised between 0.5 and 3 atm. According to an embodiment of the invention, the pressure of step (c) is comprised between 0.5 and 1 atm, preferably between 0.6 and 0.9 atm. According to another embodiment of the invention, the pressure of step (c) is comprised between 1 and 3 atm, preferably between 1.1 and 2.5 atm. Preferably, the concentration of oxygen in the reaction mixture of step (c) is below 15 ppm, preferably comprised between 0.01 and 20 ppm, more preferably between 0.1 and 10 ppm. Preferably, step (c) is conducted until completion of the reaction, preferably for a period of time comprised between 2 and 60 minutes, more preferably between 5 and 15 minutes. According to a preferred embodiment, the method of the present invention is performed in a reaction vessel. Preferably, according to said embodiment, step (c) is performed by closing and sealing the reaction vessel to advantageously reach the above-mentioned pressure and oxygen concentration conditions. According to an embodiment, the method of the present invention further comprises a step (d) of cooling the reaction mixture obtained after step (c), preferably down to a temperature comprised between 15 and 30 °C, more preferably between 18 and 28 °C.

Preferably said step (d) is performed at a cooling rate comprised between 5 and 100 minutes, preferably between 30 and 60 minutes.

Said step (d) is preferably performed under magnetic stirring, preferably at a stirring speed between 300 and 600 rpm.

According to a preferred embodiment, the method of the present invention does not comprise employing an organic solvent and a shape-directing agent that is difficult to remove after synthesis, such as a polymer and/or a surfactant. In other words, the reaction mixture does not comprise an organic solvent and a shape-directing agent that is difficult to remove after synthesis, such as a polymer and/or a surfactant.

Common organic solvents are for example, aliphatic hydrocarbons, cyclic hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, ketones, amines, esters, alcohols, aldehydes, and ethers. Common polymers used as shape-directing agents are for example, poly(vinylpyrrolidone), sodium polyacrylate and polyethylene glycol). Common surfactants used as shape-directing agents are for example, 1 ,2- Hexadecanediol, oleylamine, hexadecylamine and cetyltrimethylammonium bromide.

According to this preferred embodiment of the invention, the presence of contaminants on the surface of the nanoparticles, which could affect the catalytic properties of the nanoparticles and/or their bio-nano interactions, is avoided. Advantageously, the present invention provides a facile method for synthesizing shaped Pt nanoparticles with octahedral and/or truncated octahedral shape (i.e. with {111} ordered surface domains) without the use of Pt seed growth nanoparticles and, preferably, without the use of a shape-directing agent that is difficult to remove after synthesis, such as polymers and/or surfactants. Furthermore, being the method of the invention a seed growth-free method, namely a method which does not include a seed-growth step wherein Pt seed growth nanoparticles are employed to allow forming larger Pt nanoparticles, the synthesis steps are reduced to a simple single- step. This allows an easier and more eco-friendly production of the desired shaped ultra-small nanoparticles. In addition, as already stated before, according to a preferred embodiment, the method according to the present invention does not employ an organic solvent and is therefore more environmentally and cost friendly that many of the existing method for nanoparticles synthesis that use organic solvents as a reaction medium. The nanoparticles which can be produced according to the method of the present invention comprise a high percentage of shaped Pt nanoparticles with {111} facets, therefore Pt nanoparticles having octahedral shape and/or truncated octahedral shape.

Preferably, said shaped Pt nanoparticles obtainable with the method of the present invention have average size below 4 nm, octahedral and/or truncated octahedral shape and a percentage of {111} surface domains comprised between 15 and 35%, preferably between 18 and 24 %.

Without wishing to be bound to a particular theory, the method according to the present invention, which is thought to proceed under kinetic control, advantageously produces Pt nanoparticles with octahedral shape which leads the desired predominance of {111} facets whilst avoiding the occurrence of an uniform distribution of different facets as in spherical nanoparticles.

Advantageously, due to the pressure and oxygen concentration conditions of the above-described step (c), to the use of a first and a second reducing agent in step (b), and to the reaction temperature of step (a), the method according to the present invention allows obtaining Pt nanoparticles, with octahedral and/or truncated octahedral shape, with a percentage of {111} surface domains comprised between 15 and 35%, preferably between 18 and 24 % and with an average size - measured with transmission electron microscopy - below 4 nm, preferably below 3.8 nm, more preferably below 3.3 nm.

Said nanoparticles of said average size can be also defined, for the purpose of the present invention, as “ultra-small” nanoparticles.

Without wishing to be bound to a particular theory, Applicant has found that the above-mentioned advantages of the method according to the present invention, derive to the above-described particular combination of reaction temperature, reducing agents, pressure and oxygen concentration conditions which play a crucial role in the formation of the desired shaped Pt nanoparticles with prevalence of {111} surface domains with respect to {100} domains and an “ultra-small” average size below 4 nm.

As also demonstrated in the experiments reported below, performing the reaction in the same reaction conditions but without putting the reaction mixture under a pressure of between 0.5 and 3 atm, and without maintaining the reaction mixture under a reduced oxygen atmosphere, lead to the production of nanoparticles with a spherical shape that do not present a predominance of {111} surface domains.

The method according to the present invention thus advantageously allows producing nanoparticles highly beneficial for applications in fuel cells, catalysis and biology, as high prevalence of {111} surface domains with respect to uniform distribution of several domains on the surface as, for example, in spherical nanoparticles { (thus resulting in a octahedral and/or truncated octahedral shape) and lack of contaminations, strongly and positively impact on the catalytic properties and bio-nano interactions of the nanoparticles.

The present invention also relates to Pt nanoparticles having an average size - measured with transmission electron microscopy - below 4 nm, preferably below 3.8 nm, more preferably below 3.3 nm, octahedral and/or truncated octahedral shape and a percentage of {111} surface domains comprised between 15 and 35%, preferably between 18 and 24 %.

Preferably, said Pt nanoparticles are obtained with the method according to the present invention.

The present invention further relates to the Pt nanoparticles as described above for use in a diagnostic method.

Preferably said diagnostic method is an immunoassay. According to a preferred embodiment of the invention, said Pt nanoparticles for use in a diagnostic method, preferably in an immunoassay, are adsorbed to an antibody, preferably said Pt nanoparticles are passively adsorbed to an antibody.

Without wishing to be bound to a specific theory, the Applicant found that the Pt nanoparticles of the present invention having an octahedral and/or truncated octahedral shape possess superior catalytic/enzymatic ability even when bound to an antibody compared to nanoparticles with similar size but with a spherical shape (as described below in the Examples). In other words, said diagnostic method preferably comprises the step of employing the Pt nanoparticles of the present invention, preferably passively adsorbed to an antibody, and performing UV-Vis spectroscopy measurements.

EXAMPLES

Example 1 - Preparation of ultra-small Pt shaped nanoparticles The synthesis was performed in a 250 ml round bottom flask as a reaction vessel placed in a glycerol bath already at 90 °C, under magnetic stirring at around 500 rpm.

Pt nanocrystals were synthesized according to the method of the present invention by following the above-mentioned steps (a)-(d), in particular by introducing 53 mI of hexachloroplatinic acid aqueous solution 0.5 M (BioXtra grade, Sigma-Aldrich) into a 90 ml of Milli-Q water at 90 °C. After 1 minute, 2.2 ml of a solution containing 40 mM sodium citrate and 3 mM citric acid was added to the reaction vessel. The quick addition of 2 ml of 25 mM aqueous solution of sodium borohydride (NaBhU), freshly prepared, was followed by the closure of the reaction vessel to control the internal pressure and the exposure to atmospheric oxygen.

After 10 minutes with a fixed temperature, the solution was cooled to room temperature (25 °C) under magnetic stirring (500 rpm). The synthesis does not require the use of metallic seeds.

Example 2 - Preparation of Pt nanoparticles according to the prior art (comparative example)

The synthesis was repeated as described in as first embodiment of Moglianetti et al. “Citrate-coated, size tunable octahedral platinum nanocrystals: a novel route for advanced electrocatalysf , ACS Applied Materials and interfaces, 2018.

55 mI of hexachloroplatinic acid aqueous solution 0.5 M (BioXtra grade, Sigma-Aldrich) were introduced into a 90 ml of Milli-Q water at boiling temperature. After 1 min, 2.2 ml_ of 35 mM sodium citrate and 3 mM citric acid aqueous solution was added, immediately followed by a quick addition of 1.1 ml_ of 22 mM aqueous solution of NaBhU, just dissolved. After 10 minutes, the solution was cooled to room temperature.

This synthesis has been performed with an open reflux setup, namely without applying a pressure of between 0.5 and 3 atm and without maintaining the reaction mixture under a reduced oxygen atmosphere, as instead in the case of the method of the present invention wherein a closed reaction vessel has been employed.

Example 3 - Preparation of Pt nanoparticles according to the prior art (comparative example)

The synthesis was repeated as described in as second embodiment of Moglianetti et al. “Citrate-coated, size tunable octahedral platinum nanocrystals: a novel route for advanced electrocatalysf , ACS Applied Materials and interfaces, 2018.

55 mI_ of hexachloroplatinic acid aqueous solution (0.5 M) was added to 90 ml_ of Milli-Q water at boiling temperature. After 1 min, 2.2 ml_ of 35 mM sodium citrate and 3 mM citric acid aqueous solution was added. The solution is then immersed in ice to further decrease the temperature and, hence, slow down the growth. This step is followed by the addition of a NaBhU aqueous solution. The final solution is sealed within a glass container and then abruptly transferred to a silicon oil bath already at 100°C to obtain a fast increase of temperature up to boiling point in a closed vessel leading to a quick reduction of the Pt ions and, hence, smaller particles.

Example 4 - Physical-Chemical Analysis

The percentage of the {111} ordered domains has been determine both for the nanoparticles obtained according to Example 1 (i.e. method of the present invention) and for those obtained according to Examples 2/3 (i.e. prior art method).

The results are reported in Table 1. Table 1

The % of {111} sites have been calculated using the electrochemistry setups and procedures described in Moglianetti et al. “Citrate-coated, size tunable octahedral platinum nanocrystals: a novel route for advanced electrocatalysf , ACS Applied Materials and interfaces, 2018. The adsorption of bismuth was performed in a saturated B12O3 solution + 0.5 M H2SO4 solution. After Bi was adsorbed on the electrode surface, the electrode was rinsed with ultra-pure water and immersed at 0.05 V in the characterization cell containing a 0.5 M H2SO4 solution. From the area of the peak in the positive sweep scan, the charge related to the Bi desorption (qBi) can be calculated (equation 1 , Figure 8):

(1) 0.017 0.34

From this charge value and using the equation (2) extracted from literature (Moglianetti et al. as cited above):

(2) q_Bt = 0.64q _lir¾ the charge of the {111} terraces (qcin)¹) can be obtained. After that and from the value of 240 pCcrrr² for a Pt{111} (100% {111} sites), the percentage of {111} terrace sites can be obtained using the equations (3) and (4) (wherein, for the scope of the calculation, the average size of the octahedral Pt nanoparticles of Example 1 has been assumed to be equal to 3 nm):

Bright-field transmission electron microscopy (BF-TEM) images have been recorded using JEOL JEM 1011 transmission electron microscope in order to show shape and size of the Pt nanoparticles obtained according to the method of the present invention (Figure 1). In Figure 2, the size distribution of the ultra-small octahedral Pt nanoparticles obtained with the method of the present invention (according to Example 1), is shown: the average size being 2.8 ± 0.5 nm (see Table 1 above).

Said size distribution has been evaluated using ImageJ software. High-angle annular dark-field scanning transmission electron microscopy (FIAADF STEM) images have been recorded to further characterize the shape of the Pt nanocrystals obtained with the method according to the present invention (Figure 3).

Said high-angle annular dark-field scanning transmission electron microscopy (FIAADF STEM) images have been recorded by using FEI Tecnai G2 F20 TWIN microscope, with a Schottky emitter and operated at 200 kV.

More specifically, the average (lateral) size of the nanoparticles was obtained by manual imposing a threshold on the FIAADF-STEM images followed by automatic measurement of the Feret's diameter using the ImageJ software. More than 300 particles were considered for the distribution. In the context of the present invention, the term “Feret’s diameter” is substituted with the more generic “average size” or “average lateral size” to indicate the dimension of the nanoparticles with the same meaning used by Xia et al. in one of the most important reviews to define the dimension of shaped nanoparticles (Shape-Controlled Synthesis of Colloidal Metal Nanocrystals: Thermodynamic versus Kinetic Products, Authors: Younan Xia, Xiaohu Xia, and Hsin-Chieh Peng, DOI: 10.1021 /jacs.5b04641 , J. Am. Chem. Soc. 2015, 137, 7947-7966).

In addition, high-resolution transmission electron microscopy (HR-TEM) images have been recorded to show the shape and facets of the ultra small octahedral platinum nanoparticles obtained with the method of the present invention (Figure 4).

High-resolution transmission electron microscopy (FIR-TEM) images have been recorded to have an in-depth characterization of the surface structure. HR-TEM images have been acquired using a Cs-image corrected JEOL JEM-2200FS TEM (Schottky emitter, operated at 200 kV). The fine control over temperature and heating rate in a closed vessel plays a crucial role in the formation of the ultra-small Pt shaped nanocrystals. The same reaction conditions, in an open reflux setup, according to the teachings of the prior art (Example 2), do not produce preferentially shaped nanocrystals, but spherical nanoparticles, as shown in Figure 5.

In the case of the prior art method, the step described in Example 2 is the first step of a two-step seed-growth based synthesis. In that case, in order to obtain nanoparticles of octahedral shape, the spherical nanoparticles obtained with the steps described in Example 2 must undergo a further “growth” step (said spherical nanoparticles can be intended as “nanoparticle seeds” in the overall synthesis described by the prior art paper Moglianetti et al. “Citrate-coated, size tunable octahedral platinum nanocrystals: a novel route for advanced electrocatalysf , ACS Applied Materials and interfaces, 2018). Furthermore, also if comparing the final octahedral nanoparticles obtained at the end the seed growth-based synthesis of said prior art method, said nanoparticles are way larger (7 nm or 18 nm if the seeds obtained as per Example 3 or 2 are respectively employed in the second growth step described in Moglianetti et al.) than those obtained with the seed growth- free method of the present invention (see Table 2 below).

Table 2

This underlines the crucial role of the synergy of the reaction parameters of the method according to the present invention in achieving the growth of octahedral shaped nanocrystals.

Bright-field transmission electron microscopy (BF-TEM) images have been recorded using JEOL JEM 1011 transmission electron microscope. The fine control over temperature and the necessity of the injection of the reducing agents at 90°C play a crucial role in the formation of the ultra small nanocrystals.

The same reaction conditions in a closed vessel but with a quick increase of the temperature from 0°C to boiling point, as described in Example 3, do not produce preferentially shaped nanocrystals, but spherical nanoparticles, Figure 6. This underlines the crucial role of the synergy of the reaction parameters in achieving the growth of octahedral nanocrystals. Example 5 - Deposition of Pt nanoparticles on a conductive substrate The Pt octahedral nanoparticles with an average size of 2.8 nm have been deposited on a conductive amorphous carbon substrate with a fast and easy deposition step involving the addition of a strong base to the solution containing the Pt nanoparticles obtained at the end of the synthesis described in Example 1.

In order to ensure the complete removal of citrate, the substrate surface is washed with ultra-pure water. As shown in Figure 7, the removal of citrate is complete and a very well dispersion of the nanoparticles on the substrate without the formation of agglomeration is achieved. The conductive amorphous carbon substrate covered with the Pt nanoparticle produced with the method of the present invention does not contain impurities (such as residual polymers and/or surfactants) and can therefore be advantageously applied in technological applications, such as in fuel cells, with improved performances.

Example 6 - Colorimetric evaluation of the enzymatic activity of Pt nanoparticles of the invention and of Pt nanoparticles according to the prior art (comparative example)

The superior catalytic/enzymatic ability of ultra-small Pt nanoparticles having an octahedral shape of the present invention (synthetized according to Example 1) compared to state-of-the-art spherical nanoparticles with similar size (synthesized following the method described in Example 3) was discovered by assessing their HRP- mimicking activity.

In order to investigate the HRP-mimicking activity, 3, 3’, 5,5’- tetramethylbenzidine (TMB) was chosen as chromogenic substrate.

The TMB oxidation reaction kinetics has been characterized by UV-Vis spectroscopy measurement (absorbance peak at 652 nm). The reaction kinetics were monitored for 300 seconds.

The HRP-like activity test was performed at room temperature under acidic pH by using 7 mM Acetate buffer at pH 4.5, 35mM TMB and 435 mM H2O2 as oxidizing agent. The concentration of the two nanomaterials with the same size was kept constant at 0.005 ppm in order to evaluate the difference in performance of the surface structures present in the case of octahedral and spherical nanoparticles.

The ultra-small Pt nanoparticles having an octahedral shape synthetized according to Example 1 outperform the spherical nanoparticles (synthetized according to Example 3). As shown in Figure 9, the absorbance at 300 seconds is almost two times higher, demonstrating a higher catalytic activity in the oxidation of TMB.

This experiment demonstrates the effect of the different shape on the physical-chemical properties of the nanoparticles.

Example 7 - Colorimetric evaluation of the enzymatic activity of Pt nanoparticles of the invention and of Pt nanoparticles according to the prior art once passively adsorbed to antibodies (comparative example)

The catalytic/enzymatic ability of ultra-small Pt nanoparticles having an octahedral shape of the present invention (synthetized according to Example 1) compared to state-of-the-art spherical nanoparticles with similar size (synthesized following the method described in Example 3) has been also evaluated after the passive adsorption of the nanomaterials to the antibodies.

In order to evaluate the different performance of Pt nanoparticles of different shape once bound to antibodies, 3,3’,5,5’-tetramethylbenzidine (TMB) was chosen as chromogenic substrate (a common substrate in enzymatic immunoassays) and FITC Mouse Anti-Fluman CD195 (BD Pharmingen) has been selected as the antibody to functionalize.

The test was performed at room temperature under acidic pH by using 7 mM Acetate buffer at pH 4.5, 35 mM TMB and 435 mM FI202 as oxidizing agent. CD195 antibodies have been functionalized with the two set of Pt nanomaterials following the protocol reported by Tam et al. for the passive adsorption/passive conjugation of citrate-coated nanoparticles to antibodies (J. Immunoassay Immunochem., 2017, 38(4), pp 355-377, doi:10.1080/15321819.2016.1269338). Extensive purification through centrifugation has been performed to remove the unbound nanomaterials. Octahedral ultra-small Pt nanocrystals bound to the antibodies give a higher signal than the spherical nanoparticles of Example 3 bound to the antibodies (Figure 10), keeping identical the concentration, the conditions and the purification steps for the passive adsorption for the two nanomaterials. The absorbance at 300 seconds is indeed two times higher, demonstrating a higher catalytic activity of the octahedral ultra small Pt nanocrystals in the oxidation of TMB even when bound to the antibodies. This experiment demonstrates that the higher performance of octahedral ultra-small Pt nanocrystals of the present invention is maintained even when bound to antibodies with an increase in the signal of more than 50% compared to the state-of-the-art spherical nanoparticles.

This discovery can be applied to improve the sensitivity of commonly used immunoassays.

Claims

1. A seed growth-free method for producing Pt nanoparticles with octahedral and/or truncated octahedral shape, having an average lateral size, measured with transmission electron microscopy, below 4 nm, comprising the steps of:

2. The method according to claim 1 , wherein said reaction mixture does not comprise an organic solvent and a shape-directing agent that is difficult to remove after synthesis, preferably a polymer and/or a surfactant.

3. The method according to claim 1 or 2, wherein said Pt precursor is selected from the group consisting of a salt, a complex salt or an acid of Pt soluble in water, preferably selected in the group consisting of: H2PtCl6, Na₂PtCI₆, K₂PtCI₆, H₆CI₂N₂Pt, PtCI₂, PtBr₂, Li₂PtCU, H₂Pt(OH)₆, Pt(N0₃)2, PtSC>4, Pt(HSC>4)₂, Pt(CIC>4)₂, hydrates therefor or a mixture of salts and/or hydrates thereof.

4. The method according to any one of the preceding claims, wherein the first reducing agent is an organic molecule having less than 8 carboxylic acids and/or carboxylate groups, and less than 6 hydroxyl groups, preferably selected from the group consisting of sodium citrate, citric acid, formic acid, pyruvic acid, oxalic acid, maleic acid, organic molecules which are formed during the oxidation process of sodium citrate such as acetone and formaldehyde, and combination thereof, more preferably a combination of sodium citrate and citric acid.

5. The method according to any one of the preceding claims, wherein the first waiting time is comprised between 0.5 and 30 minutes, preferably between 0.8 and 10 minutes.

6. The method according to any one of the preceding claims, wherein the second reducing agent is selected from the group consisting of: sodium borohydride, lithium borohydride, lithium aluminum borohydride, lithium triethylborohydride, diisobutylaluminium hydride and combinations thereof, preferably sodium borohydride.

7. The method according to any one of the preceding claims, wherein the second waiting time is comprised between 0.1 and 20 seconds, preferably between 2 and 5 seconds.

8. The method according to any one of the preceding claims, wherein the pressure of step (c) is comprised between 0.6 and 0.9 atm or between 1.1 and 2.5 atm.

9. The method according to any one of the preceding claims, wherein the concentration of oxygen in the reaction mixture of step (c) is below 15 ppm, preferably comprised between 0.01 and 20 ppm, more preferably between 0.1 and 10 ppm.

10. The method according to any one of the preceding claims wherein a reaction vessel is employed and step (c) is performed by closing and sealing said reaction vessel.

11. Pt nanoparticles having an average lateral size, measured with transmission electron microscopy, below 4 nm, preferably below 3.8 nm, more preferably below 3.3 nm, octahedral and/or truncated octahedral shape and a percentage of {111} surface domains comprised between 15 and 35%, preferably between 18 and 24 % measured according to the method as described on page 19 of the description.

12. Pt nanoparticles according to claim 11 for use in a diagnostic method.

13. Pt nanoparticles for use according to claim 12, wherein the diagnostic method is an immunoassay.

14. Pt nanoparticles for use according to claim 11 or 12, wherein the Pt nanoparticles are adsorbed on an antibody, preferably passively adsorbed.