WO2021198820A1

WO2021198820A1 - A method and kit for detecting an analyte in a sample

Info

Publication number: WO2021198820A1
Application number: PCT/IB2021/052039
Authority: WO
Inventors: Pier Paolo Pompa; Poalo DONATI
Original assignee: Fondazione Istituto Italiano Di Tecnologia; Università Degli Studi Di Genova
Priority date: 2020-04-03
Filing date: 2021-03-11
Publication date: 2021-10-07
Also published as: IT202000007162A1

Abstract

A method for detecting an analyte in a sample, wherein the analyte is selected from hydrogen peroxide and/or at least one compound capable of generating hydrogen peroxide, the method comprising the steps of: i) providing a substrate comprising branched nanoparticles of at least one noble metal, said branched particles comprising a spherical body and at least one branch protruding from the body, wherein said branched nanoparticles are free of capping agents and surfactant agents, ii) placing said substrate in contact with: - the sample to be subjected to detection of said analyte, - at least one component selected from halides, pseudohalides, aromatic amines, 2,2'-azino-bis (3-ethylbenzothiazolin-6-sulphonic) acid, and mixtures thereof, iii) detecting a color variation of said substrate after a period of time T1, wherein said color variation of said substrate after the period of time T1 is determined by a plasmonic shift deriving from remodeling of said nanoparticles and indicates the presence of said analyte in the sample.

Description

“A method and kit for detecting an analyte in a sample”

TEXT OF THE DESCRIPTION

Field of the invention

The present description relates to methods for determining an analyte in a sample. In particular, the description refers to methods and kits for detecting hydrogen peroxide or at least one compound capable of generating hydrogen peroxide in a sample.

Background of the invention

The recognition and dosage of bio-markers for prevention, early diagnosis and health assessment is a topic of worldwide interest. Current commonly accessible techniques for analyzing these markers involve invasive sampling, such as blood withdrawal, by specialized employees in equipped facilities, such as clinics or appropriately set up pharmacies. Parallel to the emergence of telemedicine and the refinement of therapeutic approaches, the need for autonomous remote monitoring and analysis is increasing, with the aim of obtaining results in real time. The growing need to constantly monitor certain parameters or perform low-cost prescreening tests has led to the rapid spread of remote or point-of-care (POC) tests. These POC techniques have short execution times, do not require instrumentation and are not invasive, to the point that they are also applied in self-monitoring/self-analysis. In this context, methods based on non-invasive sampling of diagnostic fluids are of particular interest. Saliva is a useful diagnostic tool as it is easy to sample, rich in composition, and many of its constituents are correlated in concentration with blood values.

A target of great interest in this area is hydrogen peroxide (H₂O₂), which plays an important role both as first indicator of the state of oxidative stress and as a second messenger in the context of enzymatic reactions, for the recognition and dosage of biomolecular targets or environmental pollutants. Quantifying the peroxide can, therefore, be intended both to assess the ROS load that affects the body, but also as a stoichiometric product of the conversion of indicators of pathological or physiological states such as antigens, carbohydrates, lipids or circulating metabolites. These targets can be quantified thanks to reactions catalyzed by oxidase-type enzymes which, in addition to the oxidized form of the target analyte, provide H₂O₂ as a secondary product starting from H₂O and O₂.

The POC methods developed for these applications are mainly based on the color variation of solutions or solid supports. The basis of these technologies is the use of nanoparticles, typically of noble metals.

It is known that gold nanoparticles have optical properties in terms of localized surface plasmon resonance (LSPR), closely related to their shape and size, so that to date, a variety of shapes with plasmonic properties can be synthesized, which are unique for each type of particle. Processes that modify the morphology, as happens for erosion (or etching), have a strong impact on the plasmonic properties of these particles, which results in a change in the macroscopic properties (in terms of color of suspensions comprising these nanoparticles). The erosion of the nanoparticle, in fact, causes a reduction in its size and, consequently, a reduction in the intensity of the characteristic plasmonic peak; this, moreover, may also undergo significant shifts in terms of wavelength during this process.

This phenomenon can be exploited in POC technologies if the erosion is controlled and related to the presence of an analyte of interest in solution. As mentioned, an analyte of particular interest is hydrogen peroxide; this compound is able to promote the erosion of metal nanoparticles (nanosensors) in solution, allowing a morphological and/or dimensional change of the nanoparticles, and a consequent variation in the intensity and/or position of the localized surface plasmon resonance peak of these nanoparticles. Currently known methods of remodeling gold nanoparticles through oxidative erosion phenomena may involve the use of nanoparticles coated and protected by one or more capping agents; these methods may have the disadvantage whereby the remodeling phenomenon occurs in very long time intervals, of a few hours or days. Furthermore, in addition to a morphological change of the particles, there may be a significant reduction in their size, which does not allow the appreciation of a visual colorimetric variation, but involves the need to conduct specific instrumental analyses. Other methods may include the possibility of detecting the presence of hydrogen peroxide in a sample by means of colorimetric variation induced by the erosion of nanoparticles. Nanoparticles that can be used in these methods can be synthesized in the presence of silver and be coated with an organic capping agent, such as polyvinylpyrrolidone (PVP). Examples of the use of noble metal nanoparticles coated with capping agents or surfactants are described, for example, in the following documents. The scientific publication by Laura Rodriguez et al. (2011) “ Reshaping and LSPR tuning of Au nanostars in the presence of CTAB ”, Journal of Materials Chemistry, 21, 7405-7409, describes colloidal gold nanoparticles coated with PVP as a capping agent, wherein the addition of hexadecyltrimethylammonium bromide (CTAB) causes a structural modification of the nanoparticles. The document CN 109 692 972 A 1 describes platinum-palladium nanoparticles (“ PtPd nanoflower ”) produced in a solution of hydrochloric acid and F127, and their use in a catalytic reaction for detecting hydrogen peroxide in a sample. The document by Ying Ma et al. (2016) “ Facile synthesis of hierarchical gold nanostructures and their catalytic application” , Nanotechnology 27, 32:1-7 describes gold nanoparticles coated with the PNAAN polymer as a capping agent and their use, for example, in catalytic reactions. These methods may require long periods of time for colorimetric detection and the use of reagents, for example, strong bases such as sodium hydroxide, which in light of the high corrosive power require specific protective measures for use.

Object and summary of the invention

The present invention relates to a simple, sensitive and rapid method for the colorimetric determination of an analyte in a sample.

According to the present description, this object is achieved thanks to a method having the characteristics forming the subject of the attached claims. The claims form an integral part of the disclosure provided here in relation to the described method.

The method described here allows detection of the presence of an analyte in a sample, wherein the analyte is selected from hydrogen peroxide and/or at least one compound capable of generating hydrogen peroxide, the method comprising the steps of: i) providing a substrate comprising branched nanoparticles of at least one noble metal, said branched nanoparticles comprising a spherical body and at least one branch protruding from the body, wherein said branched nanoparticles are free of capping agents and surfactant agents, ii) placing said substrate in contact with:

- the sample to be tested for detecting said analyte,

- at least one component selected from halides, pseudohalides, aromatic amines, 2,2'-azino-bis (3-ethylbenzothiazolin-6-sulphonic) acid, and mixtures thereof, iii) detecting a color variation of said substrate after a period of time Ti, wherein said color variation of said substrate after the period of time Ti is determined by a plasmonic shift deriving from remodeling of said nanoparticles, and indicates the presence of said analyte in the sample.

Preferably, when the analyte is at least one compound capable of generating hydrogen peroxide, the substrate is also placed in contact with at least one oxidase enzyme, specific for said compound.

In one or more embodiments, said substrate may be a liquid substrate or a solid substrate.

Preferably, the sample is a liquid sample.

The present description also provides a kit for detecting an analyte in a sample, wherein the analyte is selected from hydrogen peroxide and at least one compound capable of generating hydrogen peroxide, the kit comprising:

- a substrate comprising branched nanoparticles of at least one noble metal, said branched nanoparticles comprising a spherical body and at least one branch protruding from the body, said branched nanoparticles being free of capping agents and surfactant agents,

- at least one compound selected from halides, pseudohalides, aromatic amines, 2,2'-azino-bis (3-ethylbenzothiazolin-6-sulphonic) acid, and mixtures thereof,

- optionally at least one oxidase enzyme specific for said compound capable of generating hydrogen peroxide and/or a buffer solution containing at least one acid.

The kit may also comprise a buffer solution comprising at least one acid.

The capping agents and surfactant agents of which the nanoparticles are devoid of can be selected from hexadecyltrimethylammonium bromide (CTAB), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), bovine serum albumin (BSA), silver nitrate.

Advantageously, the color detection of the substrate comprising the nanoparticles can be carried out by visual inspection.

Brief description of the figures

The method will now be described in detail with reference to the attached figures, given purely by way of non-limiting example, wherein:

- Figure 1 represents an enzymatic catalysis mechanism with the formation of hydrogen peroxide as a secondary product; - Figure 2 represents an example of conducting the method subject of the present description wherein the substrate comprising the nanoparticles is a liquid substrate placed in contact with the sample and with a halide salt (represented by a drop); when hydrogen peroxide is present in the sample, the erosion agent is formed, such as hypobromous acid, indicated by the generic abbreviation HXO;

- Figure 3 represents a diagram of the method of the present description according to an embodiment wherein the substrate comprising the nanoparticles is a solid substrate placed in contact with the sample and the compound capable of generating the erosion agent (represented by a drop);

- Figure 4 relates to transmission electron microscope (TEM) images of branched gold nanoparticles observed before (left) and after (right) conducting the method on a sample containing ImM H2O2. By demonstrating that the colorimetric response is due only to the uncatalyzed erosion and remodeling process, it can be observed that the process is limited to the surface of the nanoparticles only, in particular to the branches; no decrease in size of the nanoparticles is observed;

- Figure 5 relates to a comparative test wherein three different types of nanoparticles were compared. Under the conditions of the method described, only the branched nanoparticles (nanostars) respond to the presence of hydrogen peroxide with a significant plasmonic shift in the 5 minutes envisaged by the test,

- Figure 6 is an image that relates to a comparison experiment of a method carried out using nanoparticles of different shapes: from the top in the first row (wells A1-A4) branched nanoparticles; below (wells B1-B4) nanobars and, finally (wells C1-C4) nanospheres,

- Figure 7 represents the plasmonic shift (from right to left) during conducting of the method described, originating from the enzymatic oxidation of glucose present in a saliva sample. In just 5 minutes, the nanoparticles show a significant change in color without reducing the intensity of the signal. The erosion-remodeling mechanism leads to nanoparticles having a high molar extinction coefficient allowing a clear visualization of the result;

- Figure 8 represents the plasmonic shift of a sample wherein modeling of the nanoparticles does not take place. The erosion of the nanoparticles alone causes a reduction in optical intensity, while in the operating conditions of the method subject of the present description there is a bi-modal mechanism that allows maintenance of a clear colorimetric response. Detailed description

In the following description, numerous specific details are provided to allow a thorough understanding of embodiments. The embodiments can be put into practice without one or more of the specific details or with other methods, components, materials etc. In other cases, well-known structures, materials or operations are not shown or described in detail to avoid confusing aspects of the embodiments.

Reference throughout the present disclosure to “one embodiment” or “an embodiment” indicates that a particular aspect, structure or characteristic described with reference to the embodiment is included in at least one embodiment. Thus, forms of the expressions “in one embodiment” or “in an embodiment” at various points throughout the present description are not necessarily all referring to the same embodiment. Moreover, the particular aspects, structures or characteristics can be combined in any convenient way in one or more embodiments. The titles provided in this description are for convenience only and do not interpret the scope or object of the embodiments.

The present description relates to a rapid method for the colorimetric determination of an analyte selected from hydrogen peroxide or a compound capable of generating hydrogen peroxide. The method exploits a selective and exclusive erosion phenomenon of branched nanoparticles, preferably gold and free of capping agents combined with remodeling of these nanoparticles. The method allows determining the presence of an analyte on the basis of a colorimetric variation appreciable to the naked eye, without the need to use specific laboratory instruments. The method has the advantages of being economical, having short execution times, of not requiring protection devices for its execution and is, therefore, suitable as a remote or point-of-care (POC) test.

The description provides a method for detecting an analyte in a sample, wherein the analyte is selected from hydrogen peroxide and at least one compound capable of generating hydrogen peroxide, the method comprises the steps of: i) providing a substrate comprising branched nanoparticles of at least one noble metal, said branched nanoparticles comprising a spherical body and at least one branch protruding from the body, wherein said branched nanoparticles are free of capping agents and/or surfactant agents, ii) placing said substrate in contact with: - the sample to be tested for detecting said analyte,

- at least one component selected from halides, pseudohalides, aromatic amines, 2,2'-azino-bis (3-ethylbenzothiazolin-6-sulphonic) acid, and mixtures thereof, iii) detecting a color variation of said substrate after a period of time Ti, wherein said color variation of said substrate after said period of time Ti is determined by a plasmonic shift deriving from remodeling of said nanoparticles, and indicates the presence of said analyte in the sample.

The liquid substrate can, for example, be an aqueous solution comprising the branched nanoparticles.

The solid substrate may be a substrate of a material capable of retaining the nanoparticles. The solid substrate may comprise or consist of synthetic or natural polymeric material. The material can be selected from the group consisting of polyamides (Nylon), Polyvinylidene fluoride (PVDF), cellulose derivatives, preferably cellulose acetate, cellulose esters, nitrocellulose. Furthermore, the solid substrate may have a thickness between 10 pm and 5 mm, preferably between 50 pm and 500 pm.

In order to provide the solid substrate comprising the nanoparticles, the solid substrate is supplemented with a solution of nanoparticles, preferably in the measure of 10-60 pL every 10 mm², for a final deposited quantity preferably between 2 · 10 ¹² and 10 ¹⁵ moles/mm². The solution comprising the nanoparticles is deposited on the substrate and kept on the substrate until completely absorbed by it. A substrate drying step follows. Likewise, optionally in a subsequent step, the substrate can also be supplemented with a solution comprising oxidase enzyme.

When the analyte to be detected is at least one compound capable of generating hydrogen peroxide, the substrate can also be placed in contact with at least one enzyme of the oxidase group.

The method allows determining - in a sample - the hydrogen peroxide or at least one compound capable of generating hydrogen peroxide, preferably in the presence of oxidase enzymes. The compound capable of generating hydrogen peroxide in the presence of oxidase enzymes is a compound comprising at least one hydroxyl group (OH-). Compounds capable of generating hydrogen peroxide can be selected from molecules of biochemical interest such as monosaccharide sugars, for example, glucose; lipids, metabolic products, for example, lactate; hormones, for example, cortisol; disaccharides, for example, sucrose and maltose, whereby an enzyme activating the (-OH) group or, in general, coupled is used. Preferably, this compound capable of generating hydrogen peroxide is glucose.

Preferably, this compound generates hydrogen peroxide by means of an oxidation reaction of the hydroxyl group OH- operated by an enzyme of the oxidase group. The oxidase enzyme is an enzyme capable of reacting with the specific analyte of interest (for example, the enzyme will be glucose oxidase if the analyte of interest is glucose).

Preferably, the sample is a liquid sample.

In one or more embodiments, the sample may be selected from at least one biological liquid, water, or food products. Preferably, the biological liquid may be selected from saliva, serum, urine, sweat.

When the sample is a water sample, it can be selected from surface rainwater, groundwater, water derived from rivers, or from streams. In the case of a sample derived from foodstuff material, this sample can be selected from beverages, infusions, soft drinks, sports or energy drinks. These samples can be used in the described method either as extracts or concentrates, and either homogenized or clarified. These samples may come from solid samples such as raw materials or processed products from the agro-food industry.

In one or more embodiments, the sample can be obtained by means of sampling techniques known in the art and maintained, if subjected to storage, at an appropriate temperature. For example, with reference to a sample of river water, common sampling techniques can be used for analyzing water and storing the sample at a temperature between 4°C and 40°C, preferably between 10°C and 30°C.

In the case of a biological sample, it can be subjected to the method at the same time as the sampling, but also following suitable storage, preferably at a controlled temperature between 0°C and 4°C.

In one or more embodiments, the quantity of sample to be placed in contact with the substrate may be between 10 pL and 1000 pL, preferably between 50 pL and 500 pL.

In one or more embodiments, the at least one compound capable of generating hydrogen peroxide in the presence of oxidase enzymes may be selected from monosaccharides, preferably glucose; disaccharides, preferably sucrose and maltose; lipids, preferably cholesterol; carboxylic acids, preferably lactic acid and derivatives; hormones, preferably cortisol.

Branched nanoparticles of at least one noble metal may be selected from branched gold nanoparticles and branched silver nanoparticles.

These branched nanoparticles - also referred to in the present description as star-shaped nanoparticles or star-like nanoparticles or nanostars - are preferably branched gold nanoparticles. The term “branched nanoparticles” is intended to indicate nanoparticles comprising a spherical body and at least one branch protruding from the body, preferably conical monocrystalline. Preferably, these nanoparticles comprise a plurality of branches (or protuberances). The length of each branch protruding from the spherical body is less than the radius length of the spherical body.

The nanoparticles usable in the method described do not have capping agents on the surface, such as cytotoxic capping agents, preferably selected from organic thiol molecules, surfactants, including hexadecyltrimethylammonium bromide (CTAB), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), bovine serum albumin (BSA), silver nitrate.

In one or more embodiments, said branched nanoparticles have a diameter between 1 nm and 200 nm, preferably between 15 nm and 100 nm, more preferably between 20 nm and 80 nm. The indicated value of the diameter refers to the entire particle, including the body and branches.

In one or more embodiments, said branched nanoparticles have a spherical body and branches protruding from said body. The average length of the branches protruding from the body can be between 4 nm and 60 nm. These nanoparticles guarantee a localized surface plasmon resonance (LSPR) with a wavelength (lhihc) between 560 nm and 700 nm.

These branched nanoparticles can be made by the method described, for example, in the document W02012/077043A2.

The liquid substrate may comprise branched nanoparticles in a concentration ranging from 1 fM to 100 nM, preferably between 0.01 nM and 2 nM. This concentration is expressed as the number of particles per liter of solution.

The solid substrate may comprise a quantity of branched nanoparticles ranging from 2 · 10¹² and 10¹⁵ moles/mm².

In one or more embodiments, the method may envisage placing the substrate comprising the nanoparticles in contact with a buffer solution containing at least one acid. This acid can be selected from the group consisting of citric acid, phosphoric acid, 2-(N-morpholino)-ethanesulfonic acid (MES), acetic acid, relative salts, preferably alkaline salts of sodium or potassium. This buffer solution may contain at least one acid in a concentration ranging from 1 mM to 100 mM, preferably equal to 20 mM. In one or more embodiments, the pH of the substrate also placed in contact with a buffer solution containing at least one acid may be between 4 and 7, preferably between 4.5 and 6.

The at least one component selected from halides, pseudohalides, aromatic amines, 2,2'-azino-bis (3-ethylbenzothiazolin-6-sulphonic) acid, and mixtures thereof react to determine the formation of an erosion agent when the sample comprises hydrogen peroxide or when hydrogen peroxide forms in the sample. Usable halides and pseudohalides are fluorides, bromides, chlorides, iodides, thiocyanates (e.g., thiocyanate ion (SCN-)). Halides and pseudohalides can be used in the form of halide salts. Preferably, the halide is a bromide, more preferably potassium bromide. Aromatic amines comprise 3, 3', 5,5'- tetramethylbenzidine (TMB) Another usable compound is 2,2'-azino-bis(3- ethylbenzothiazolin-6-sulphonic acid) or ABTS.

These components can be contained in an aqueous solution. These components are able to react with the hydrogen peroxide present in the sample giving rise to a reaction product, also defined here as an “erosion agent”. This component can be used in a concentration between 1 mM and 5 M. When used in the liquid substrate, this component can be preferably used in a concentration ranging from 1 mM to 200 mM, more preferably between 1 mM and 50 mM.

When the analyte of interest is a compound capable of generating hydrogen peroxide in the presence of oxidase enzymes, the substrate is also placed in contact with at least one oxidase enzyme, specific for this compound. This enzyme may be selected, for example, from glucose oxidase, lactate oxidase, cholesterol oxidase. When the analyte of interest is glucose, the enzyme is glucose oxidase.

In one or more embodiments, the enzyme may be contained in the solid substrate comprising the branched nanoparticles; the enzyme can also be contained in the liquid substrate comprising the branched nanoparticles.

In one or more embodiments, the enzyme may be placed in contact with the substrate in step ii) simultaneously with the sample and at least one compound capable of generating the erosion agent. This enzyme can be placed in contact with the substrate in step ii) simultaneously with the sample and the at least one compound capable of generating the erosion agent, for example, in the form of an aqueous solution wherein the enzyme is present in a concentration of up to at 2 mg/mL.

The concentration of the enzyme may be between 1 and 300 enzyme units (U)/mL, preferably between 1.5 and 4 U/mL when used in the liquid substrate.

In one or more embodiments, when the substrate is a liquid substrate, the method may also comprise a step of stirring said substrate after step ii) or rather, after it has been placed in contact with the sample, the at least one compound capable of generating the erosion agent and optionally the oxidase enzyme. This step may allow a homogeneous suspension to be obtained, and can be carried out by using a stirrer, or by manual shaking or by means of a disposable mixing device. This stirring step may be carried out for a period of time ranging from 10 seconds to 20 seconds, preferably 20 seconds.

Following a period of time Ti, the detection of a color variation of the substrate comprising the branched nanoparticles may be conducted by visual inspection and allows the presence of the analyte in the sample to be determined. In particular, a variation in the color of said substrate after the time Ti indicates the presence of the analyte in the sample.

The time period Ti can range from 3 minutes to 20 minutes.

When the analyte of interest is hydrogen peroxide, the time period T i may preferably be comprised between 4 minutes and 7 minutes, more preferably 5 minutes.

When the analyte of interest is a compound capable of generating hydrogen peroxide, preferably in the presence of an oxidase enzyme, the time T i may be between 5 minutes and 20 minutes, preferably 15 minutes.

During the time period Ti, and as illustrated in the example of Figure 2, the hydrogen peroxide present in the sample reacts with the halide and causes the formation of a compound (or erosion agent) that erodes the noble metal atoms (e.g. gold) that are present on the branch tips of the nanoparticles.

In the example illustrated in Figure 2, the nanoparticles are contained in a liquid substrate, for example, an aqueous solution. The color variation of the aqueous solution after the time period Ti indicates the presence of the analyte in the sample.

Figure 3 illustrates a similar example, wherein the substrate comprising the branched nanoparticles is a solid substrate, for example, a polyamide disc. The substrate is placed in contact with the sample and with the compound capable of generating the erosion agent (indicated in the Figure with a drop). A variation in the color of the substrate indicates the presence of the analyte in the sample.

In the method subject of the present description, an erosion phenomenon of the nanoparticles occurs in the presence of hydrogen peroxide accompanied by a real and actual remodeling of the nanoparticles according to an Ostwald ripening principle that occurs after erosion. This phenomenon occurs on nanoparticles that do not have capping agents, such as for example CTAB.

After the atoms at the tips have been eroded by the erosion agent, they are redistributed onto the surface of the nanoparticles according to two complementary reactions:

Reduction: H2O2 + 2AuBr₂ = 2Au + 2H⁺ + 4Br + O2 Oxidation: H2O2 + 2H⁺ + 2Au + 4Br = 2AuBr₂ + 2FhO

The atoms of the noble metal, for example, the gold atoms, are redistributed onto the surface of the nanoparticles themselves, causing a morphological variation of the nanoparticles. The branched nanoparticles assume a spherical morphology but do not undergo variations in their size. The spherical nanoparticles thus obtained have dimensions comparable to the dimensions of the starting branched nanoparticles, as shown in Figure 4, with a considerable molar extinction coefficient. This morphological variation not associated with a dimensional variation allows a shift of the localized surface plasmon resonance peak. As shown in Figures 5 and 7, the peak detected as a result of the morphological variation of the branched nanoparticles has an intensity comparable to that of the peak detected before this variation.

The shift of the localized surface plasmon resonance peak that occurs by carrying out the steps of the described method settles at 640/610 nm of the initial wavelength value at 530/570 nm after about 300 seconds, causing a change in the color of the suspension from blue to purple to red/fuchsia color.

Advantageously, detection of the color variation can be carried out by visual inspection.

The method subject of the present description allows determining the hydrogen peroxide in samples of different nature, such as biological fluids, through the use of a colorimetric nanosensor (branched nanoparticles), based on the shift of the plasmon band of branched gold nanoparticles, induced by morphological changes of these in the presence of hydrogen peroxide.

In particular, the method is based on the phenomenon of erosion and remodeling of branched nanoparticles, without loss of intensity of the plasmon band (thanks to a phenomenon of global remodeling, not pure corrosion), with the possibility of inducing this change only through the corrosion of surface nanostructures of small dimensions (a few nanometers thick) and very limited total mass (the surface cusps of the branches). This phenomenon makes it possible to induce an obvious color variation in very short time intervals (in the order of a few minutes, for example, 5 minutes). With the same amount of corrosion and, therefore, with the same amount of hydrogen peroxide contained in the sample, moreover, the use of branched nanoparticles causes a significantly more evident plasmonic shift than that observed using nanoparticles of different shapes (such as nanobars or nanospheres). This results in a color variation appreciable to the naked eye, with the advantage, therefore, of being able to carry out the method without requiring laboratory equipment. The method, which can be easily carried out even by untrained users, is therefore suitable for use as a remote or point-of- care (POC) test.

For example, corrosion in the order of 10% of the total mass of gold involves a practically complete remodeling into a spherical structure in the case of branched nanoparticles, with full colorimetric shift. In the case of nanobars or nanospheres, there is only a slight decrease in intensity (of about 10%) accompanied by a slight colorimetric change; in the specific case of nanospheres (unbranched nanoparticles) and nanobars, the colorimetric variation is not detectable to the naked eye.

The branched particles used as nanosensors in the method described have a characteristic plasmonic peak in the visible range due to the presence of branches with surface cusps of a few nanometers in size, which determine their wavelength (color) and intensity. These nanoparticles have a high surface energy, which makes them unstable and easily erodible; moreover, the mass of the surface cusps is significantly lower than the mass exposed to erosion in the case of nanobars or nanospheres (without branches), so that a colorimetric response can be appreciated. Considering that it is the vertices of the surface cusps of the branches that confer the optical characteristics, their dissolution involves a considerable shift of the plasmon towards wavelengths typical of spherical particles without branches in a response time of a few minutes, with a sudden and evident color variation.

The method described allows obtaining the results described thanks to the combination of operating conditions that allow not only erosion of the particles, but also a morphological variation of the particles, a morphological variation that does not imply significant reductions in the intensity of the plasmonic peak. A fundamental role with reference to this aspect is determined by the characteristic whereby the branched nanoparticles do not have surface coatings, such as capping agents, such as CTAB, PVP, PEG, BSA, geometry directing agents such as silver nitrate. As anticipated in the previous sections, a disadvantage deriving from the use of coated nanoparticles that are protected by one or more capping agents is related to the fact that remodeling phenomena of these nanoparticles through, for example, oxidative erosion may occur in very long time intervals, of some hours or days.

The described method may, therefore, be used to determine the hydrogen peroxide in a sample, but also compounds that generate hydrogen peroxide following an oxidation reaction.

EXAMPLES

Some non-limiting examples of different embodiments of the method subject of the present description will be provided below.

1.1 Aqueous sample containing hydrogen peroxide

The following description provides an example of the method carried out using branched nanoparticles contained in a liquid solution as substrate. In particular, the nanoparticles were placed in contact with an aqueous sample containing hydrogen peroxide and potassium bromide, as for example, described below.

To an aqueous sample (500 pL) containing 1 mM hydrogen peroxide, 10% (% v/v) of an acetate buffer (sodium acetate/acetic acid) of 100 mM and pH 4.5 (CHiCOONa, Acetic acid sodium salt, Sigma- Aldrich S1429, CH₃CO₂H Glacial acetic acid CAS 64-19-7 ACS reagent, >99.7%, Sigma-Aldrich 695092) was added; then a solution comprising 10% (% v/v) branched nanoparticles (nanostars) of gold (diameter between 60 and 80 nm), (concentration equal to 0.1 nM in the final liquid solution); a halogen salt, 10% (% v/v) potassium bromide (KBr; Potassium bromide, CAS 7758-02-3, Sigma-Aldrich 221864), (concentration equal to 10 mM in the final liquid solution obtained).

The solution obtained was observed for 5 minutes, periodically recording its Ultraviolet-Visible (UV-VIS) spectra. In particular, 200 microliters of the solution obtained as described were mixed in a transparent plate with 96 wells (96 multiwell transparent flat bottom), the plate was inserted into a reader (Tecan Spark® multimode microplate reader), the absorbance was read every 15 seconds for 5 minutes at 23 °C with 4 seconds of shaking before each reading (medium amplitude).

A sample of the solution obtained was also subjected to characterization by transmission electron microscopy (TEM). In particular, 3 microliters of the sample were transferred to a special TEM grid as a support placed under vacuum for 2 hours before image acquisition with a TEM JEOL JEM-1400Plus - Analytical 120 kV microscope.

1.2 Saliva sample

Similarly, the method was tested for its ability to detect hydrogen peroxide originating as a secondary product of a specific enzyme of an analyte of interest. In the example, the presence of hydrogen peroxide produced by the enzyme glucose oxidase was detected. In this case, the method made it possible to detect the presence of dissolved glucose in a saliva sample.

For the experimental test, the saliva sample was adulterated with glucose (Glucose Standard Solution, 1 mg/mL, Sigma- Aldrich G6918) to a final concentration of 3 mg/dL.

To 100 pL of this saliva sample, the following were added in sequence: 10% (% v/v) of 100 mM pH 4.5 acetate buffer containing an oxidase enzyme equal to 10 u/mL, 10% (% v/v) of gold nanoparticles contained in an aqueous liquid solution (up to a concentration of 0.1 nM in the final solution) and 10% (% v/v) of a halogen salt, such as potassium bromide (KBr) up to a concentration of 500 mM in the final solution. The obtained solution is kept at 37°C, recording the UV-VIS spectra for 5 minutes, as described in the previous section.

1.3 Comparison samples

The operating conditions described in Example 1.1 were used to conduct the method on aqueous comparison samples containing 1 mM hydrogen peroxide (H2O2). For the comparison samples, the method was carried out using spherical (no branching) or bar- shaped (nanobars, nanorods) gold nanoparticles. These nanoparticles have a diameter greater than about 50 nm with an aspect ratio of about 2.7.

Furthermore, for conducting colorimetric tests, hydrogen peroxide was used in the samples in three different concentrations: 1 mM, 1.5 mM and 2 mM. Control samples without hydrogen peroxide were also used.

1.4 Analysis of the localized surface plasmon resonance peak conducted with spherical nanoparticles without branching.

To 100 pL of a saliva sample the following were added in sequence: 10% (% v/v) of 2M hydrochloric acid (Hydrochloric acid, 36.5-38.0%, BioReagent, for molecular biology CAS 7647-01-0 Sigma-Aldrich H1758) containing an oxidase enzyme equal to 10 u/mL, a 10% (% v/v) solution comprising spherical, non- branched gold nanoparticles (diameter: 35 nm) up to a concentration of 0.05 nM and 10% (% v/v) of a halogen salt, potassium bromide (KBr) up to a concentration of 50 mM. The sample was kept at 37°C and the UV-VIS spectra were recorded as described in the previous sections.

1.5 Saliva sample containing glucose

The following description provides an example of the method carried out using branched nanoparticles contained in a solid substrate, or rather, a polyamide disc, as a substrate. The substrate also comprises the oxidase enzyme (glucose oxidase). The solid substrate containing the nanoparticles and the enzyme was placed in contact with a sample of saliva supplemented with glucose and potassium bromide, as described below.

A solid substrate of polyamide (Whatman® nylon membranes) with a thickness of between 100 pm and 200 pm and 13 mm in diameter was enriched with a solution of branched gold nanoparticles, in particular with 400 pL of a solution of nanoparticles with a concentration between 40-50 pM. The nanoparticles have a diameter of approximately 60 nm. The solid substrate, in particular, is supplemented with a solution of nanoparticles in the measure of 10- 60 pL every 10 mm² for a final deposited quantity between 2 · 10¹² and 10¹⁵ moles/mm².

The solution comprising the nanoparticles was deposited on the substrate by placing the filter on a holder and leaving the solution in contact with the substrate until completely absorbed.

The enriched substrate was dried under high vacuum and stored in low humidity conditions. In a second step, the substrate was enriched with oxidase enzymes (glucose oxidase) by depositing 50 pL of a solution comprising the oxidase enzyme in an amount equal to 1 mg/mL.

A known volume of saliva (100 pL) was supplemented with a standard solution of D(+)Glucose in order to make it positive for the concentration of interest (1.5 mg/dL). The sample was then supplemented with 40 pL of 5 M potassium bromide (KBr). The obtained solution was deposited on the substrate. In 10 minutes, the enzyme converted the biological molecule of interest (glucose) into hydrogen peroxide, which in the presence of potassium bromide triggered the remodeling of the nanoparticles. This resulted in a color variation of the substrate, from a blue color to a purple/red color, visible to the naked eye. The speed and intensity of the color variation may be correlated to the analyte concentration. The method may also favor being subjected to semi-quantitative applications to monitor the quantity beyond the presence of the target.

RESULTS

Figure 4 is an image obtained with a transmission electron microscope that shows the morphological evolution of the branched gold nanoparticles used in the method of the example in section 1.2. The images show a significant morphological change of the nanoparticles that, from a branched, star-shaped morphology, take on a spherical morphology. On the contrary, the diameter of the body of the particles does not undergo significant variations; the diameter detected before and after the time period Ti is between 80 nm and 100 nm (Figure 4). A similar result was observed for the nanoparticles used in the method conducted as in Example 1.1. Biological fluids, such as saliva in the example in question, have proteins that may have a protective/stabilizing effect, consequently increasing the sensitivity of the method. The TEM images of Figure 4 show the morphological modification of branched nanoparticles relative to the saliva sample described in section 1.2.

Figure 5, relating to example 1.3, shows that the method conducted using branched nanoparticles is the most sensitive, wherein the shift of the plasmonic peak is observed. This shift, monitored for a total of 60 minutes, does not occur when the method is carried out with spherical nanoparticles without branches or nanobars.

Figure 6 shows the results of a colorimetric analysis conducted downstream of methods conducted using branched nanoparticles and comparison nanoparticles, as described in example 1.3. The samples contained in the wells of the row identified with the letter A are the samples containing branched nanoparticles according to the method of the invention (in particular, A1 corresponds to the control sample without hydrogen peroxide, A2, A3, A4 correspond to samples with hydrogen peroxide in concentrations of 1 mM, 1.5 mM and 2 mM, respectively). The samples contained in the wells of the row identified with the letter B are the samples containing nanobars (in particular, B 1 corresponds to the control sample without hydrogen peroxide; B2, B3, B4 correspond to the control samples with hydrogen peroxide in the concentrations, respectively, equal to 1 mM, 1.5 mM and 2 mM). The samples contained in the wells of the row identified with the letter C are the samples containing non- branced nanospheres (in particular, Cl corresponds to the control sample without hydrogen peroxide; C2, C3, C4 correspond to the control samples with hydrogen peroxide in the concentrations, respectively, equal to 1 mM, 1.5 mM and 2 mM).

As can be appreciated from the Figure and the RGB coordinates, only the method conducted according to the invention causes a colorimetric variation of the suspension upon visual inspection, appreciable to the naked eye.

Figure 7, obtained from a saliva sample as described in section 1.2, shows how the shift of the localized surface plasmon resonance peak maintains, during the detection, an intensity comparable to the initial one, thanks to the combined action of erosion and subsequent re-deposition of the gold atoms onto the surface of the nanoparticle through an Ostwald ripening phenomenon. This phenomenon causes a remodeling of the morphology of the branched nanoparticle without a substantial dimensional change of the nanoparticle.

Figure 8 relating to the analysis of a sample subjected to a method as described in section 1.4, shows a decrease in intensity of the plasmonic peak in a test wherein erosion occurs without remodeling the surface of the nanoparticles through Ostwald ripening. In this case, the unbranched spherical nanoparticles undergo an amount of erosion that may lead, in some cases, to the halving of the diameter of the particle itself. In this case, the erosion mechanism by bromide ions and hydrogen peroxide leads to a drastic reduction in the size of the nanoparticles. This substantial change, in turn, involves a considerable decrease in the intensity of the localized surface plasmon resonance peak as the erosive process proceeds, making it necessary to carry out an instrumental colorimetric detection as the color variation is not appreciable to the naked eye.

Surprisingly, while using branched nanoparticles devoid of protective capping agents or surfactants, the described method makes use of an erosion phenomenon limited to the cusps of the branches, without affecting the body of the particles themselves. The method involves a shift of the localized surface plasmon resonance peak in terms of wavelength, with the unique characteristic that the intensity of the peak itself remains practically unchanged during the detection. Advantageously, detection of the analyte of interest (hydrogen peroxide) takes place in a very short time, in about 5 minutes, without the need to use reagents such as strong bases such as sodium hydroxide described in the prior art.

Of course, without prejudice to the principle of the invention, the details of construction and the embodiments may be widely varied, without thereby departing from the scope of the invention as defined by the claims that follow.

Claims

1. A method for detecting an analyte in a sample, wherein the analyte is selected from hydrogen peroxide and/or at least one compound capable of generating hydrogen peroxide, the method comprising the steps of: i) providing a substrate comprising branched nanoparticles of at least one noble metal, said branched nanoparticles comprising a spherical body and at least one branch protruding from the body, wherein said branched nanoparticles are free of capping agents and surfactant agents, ii) placing said substrate in contact with:

- the sample to be tested for detecting said analyte,

2. A method according to claim 1, wherein when said analyte is at least one compound capable of generating hydrogen peroxide, said substrate is also placed in contact with at least one oxidase enzyme specific for said compound, preferably selected from the group consisting of glucose oxidase, lactate oxidase, cholesterol oxidase.

3. A method according to claim 1 or claim 2, wherein the at least one compound capable of generating hydrogen peroxide is selected from monosaccharides, preferably glucose; disaccharides, preferably sucrose and maltose; lipids, preferably cholesterol; carboxylic acids, preferably lactic acid and derivatives; hormones, preferably cortisol.

4. A method according to any of the preceding claims, wherein said branched nanoparticles are selected from branched gold nanoparticles and/or branched silver nanoparticles.

5. A method according to any of the preceding claims, wherein the length of said at least one branch protruding from the body of the nanoparticles is between 4 nm and 60 nm.

6. A method according to any of the preceding claims, wherein said capping agents and surfactant agents are selected from hexadecyltrimethylammonium bromide (CTAB), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), bovine serum albumin (BSA), silver nitrate.

7. A method according to any of the preceding claims, wherein said halides and pseudohalides are selected from the group consisting of fluorides, bromides, chlorides, iodides, thiocyanates, salts thereof, and said aromatic amines comprise 3,3',5,5'-tetramethylbenzidine (TMB).

8. A method according to any of the preceding claims, wherein in step ii) said substrate is also placed in contact with a buffer solution containing at least one acid, preferably selected from the group consisting of citric acid, phosphoric acid, 2-(N-morpholino)-ethanesulfonic acid (MES), acetic acid, salts thereof.

9. A method according to any of the preceding claims, wherein said substrate comprising said branched nanoparticles is selected from a liquid substrate, preferably an aqueous solution, and a solid substrate, preferably a material comprising, more preferably consisting of synthetic and/or natural polymeric material.

10. A kit for detecting an analyte in a sample, wherein the analyte is hydrogen peroxide and/or at least one compound capable of generating hydrogen peroxide, the kit comprising:

- a substrate comprising branched nanoparticles of at least one noble metal, said branched nanoparticles comprising a spherical body and at least one branch protruding from the body, said branched nanoparticles being free of capping agents and/or surfactant agents,

- at least one compound selected from halides, pseudohalides, aromatic amines, 2,2'-azino-bis (3-ethylbenzothiazolin-6-sulphonic) acid, mixtures thereof,