Optical sensors based on nanostructures

Abstract

The present invention concerns an ultra-sensitive optical sensor, in a single configuration or integrable in a system of variable geometry sensors, which can be coupled to a light source and a detection device as well as to a reference chip, which is able to detect the presence and / or concentration of an analyte, using a suitable receptor layer (synthetic or natural), in a real matrix through the induction of hybrid plasmonic resonances caused by the interaction between the incident visible radiation and a nanostructured metal surface, not obtained by lithographic techniques, such as pollen-based techniques. The present invention further refers to a method for the realization of the sensor and to the uses in the application fields of analytical interest.

Description

[0001] OPTICAL SENSORS BASED ON NANOSTRUCTURES

[0002] Technical field

[0003] The present invention refers to an ultra-sensitive optical sensor, in a single configuration or integrable in a sensor system, which can be coupled to a light source and a detection device, which is able to detect the presence and / or concentration of an analyte in a real matrix (such as air, aeriforms, aerosols, aqueous solutions), through the induction of hybrid plasmonic resonances caused by the interaction between the incident visible radiation and a nanostructured metal surface based, for instance, on pollen.

[0004] Known art

[0005] The selective detection of particular analytes in different matrices is a growing demand in numerous application fields comprising, for instance, the diagnosis of tumor markers, the analysis of various emerging environmental pollutants, bioterrorism or biodefense, pandemic emergencies, personalized medicine, point-of-care testing for different analyses or pre-analyses. In such applications there is a need to use sensors with very high performance that allow the detection of infinitesimal quantities of analytes (e.g., proteins, ions, complexes, molecules), or of several molecules at the same time (in the case of arrays).

[0006] Among the optical sensors used, those in waveguide stand out, for instance in plastic optical fibers (POF) or in planar waveguide, both polymeric and inorganic (silica, quartz), to monitor the variation in the refractive index of a receptor through the physical effect of surface (SPR) or localized plasmonic resonance (LSPR), or those based on hybrid plasmonic phenomena present in periodic nanostructures made by lithographic techniques (electronic or optical).

[0007] In order to obtain an improvement of the plasmonic phenomenon and therefore of the performance of the biochemical sensor in terms of instrumental sensitivity and detection limit, it is possible to resort to the use of the so-called plasmonic metasurfaces based, for instance, on periodic or pseudo-periodic structures of nanocones, nanowires, nanostrips, nanopillars, or nanolattices, usually produced with complex fabrication techniques (e.g., electronic lithography) characterized by a high cost and long production times.

[0008] For instance, in WO 2022 / 189802 a sensor using plasmonic nanostructures to improve fluorescence excitation is described. This is therefore a fundamentally different principle from the one based on measuring the variation of the resonance wavelength.

[0009] In ARCADIO F. et al: "Biochemical sensitive exploiting plasmonic sensors based on gold nano-gratings and polymer optical fibers" PHOTONICS RESEARCH, vol. 9, no. 7 (2021 -07-01 ), XP093254963, ISSN: 2327-9125 a sensor based on artificial nanostructures made by complex electron beam lithography procedures is described. In addition, the sensitivity achieved with this sensor is poor and should be improved.

[0010] In the field of sensors, optical sensitivity is often defined as the detectable change in the signal (e.g. the change in the resonance wavelength) relative to a unit change in the refractive index. Biochemical sensitivity, on the other hand, is defined as the detectable change in the signal relative to a unit change in the concentration of the substance to be measured. Generally, in the case of optical biosensors, the detection limits related to the specific measurement of a given substance are typically in the order of nanograms / liter, often also obtaining a high selectivity / specificity towards the molecule of interest (target) which is properly a function of the receptor used (natural or synthetic).

[0011] There are application contexts wherein a very high biochemical sensitivity is required, i.e. a limit of detection much lower than that of the order of nanograms / liter, almost at the level of a single molecule (attomolar range). By way of example, these specifications are required in the measurement of some particular biomarkers, of various emerging pollutants, for the measurement of viruses or bacteria, in the quantitative measurement of certain substances useful in the so-called personalized and / or precision medicine, or in the tests on rare diseases, for the measurement of explosives and other specific hazardous substances.

[0012] As is evident from the analysis of known art, the development of optical sensors with very high sensitivity but at the same time economically accessible and easy to implement continues to be a primary necessity in the field of sensors. Although numerous steps forward have been made, the obstacle of obtaining nanostructured systems that are easy to find, easy to produce and easy to use has not yet been overcome. Added to this is the continuous search for sensor devices that are not only able to detect particularly low analytical concentrations (range of 10’18molar) but that can also perform measurements on a wide variety of analytes, even simultaneously, thanks to an intrinsic ductility and instrumental versatility.

[0013] Unless otherwise specified, the contents of this section are to be considered as an integral part of the detailed description below.

[0014] Summary of the invention

[0015] The first purpose of the present invention is the realization of an optical sensor or sensor chip as stated in claim 1 .

[0016] Other purposes of the invention are a sensor system also called equipment that comprises the sensor chip of the invention, and the process for making and using said sensor.

[0017] Yet another purpose of the invention is the method of detecting an analyte by means of the invention sensor, comprising:

[0018] • irradiating the sensor with electromagnetic radiation;

[0019] • acquiring the resulting optical spectrum;

[0020] • determining the wavelength shift of the maximum plasmonic resonance as a consequence of the binding of the analyte to the receptor, wherein wavelength shift determination allows for higher sensitivity and accuracy than plasmonic band width measurement. Further purposes will be evident from the detailed description below.

[0021] Brief description of the Figures

[0022] The invention will be described below in at least one preferred embodiment for the sole purpose of explanation and not limiting the scope of the present invention with the help of the figures in the annex, wherein:

[0023] • Figure 1a: natural nanostructure based on sunflower pollen;

[0024] • Figure 1 b: sunflower pollen opening;

[0025] • Figure 1c: immobilization of pollen (in Figure 1 b) on a transparent and guiding substrate 1000;

[0026] • Figure 1d: coating of immobilized pollen with a nanometer metal layer;

[0027] • Figure 1e: scheme of the optical sensor based on a periodic or pseudo-periodic natural structure;

[0028] • Figure 2a: scanning electron microscope (SEM) image of the optical sensor surface based on pseudo-periodic natural nanostructures;

[0029] • Figure 2b: image acquired at SEM and relating to a detail of the pseudo-periodic nano-tips present on the surface of sunflower pollen;

[0030] • Figure 2c: SEM image of a nano-urchin structure;

[0031] • Figure 2d: schematization of a nano-urchin carbon structure;

[0032] • Figures 3a-3c: SEM images of details of nano-tips on the surface of nano-urchins;

[0033] • Figure 4a: schematic view of a sensor system according to the present invention;

[0034] • Figure 4b: schematic view of an optical multisensor according to the present invention; • Figure 5a: schematic view of a possible experimental configuration of the sensor system;

[0035] • Figure 5b: schematic view of a possible experimental configuration of the sensor system;

[0036] • Figure 6a: normalized hybrid plasmonic resonance spectra of the ultrasensitive optical sensor as a function of wavelength;

[0037] • Figure 6b: comparative graph showing the absolute value of the change in the resonance wavelength towards the refractive index of the external medium for the optical sensor of the invention and for a standard comparison sensor based on a nanolattice produced by electron beam lithography (EBL);

[0038] • Figure 7: hybrid plasmonic resonance spectra of the optical sensor as a function of wavelength, normalized with the spectrum acquired by the reference chip and acquired at different concentrations of estradiol;

[0039] • Figure 8: graph of the absolute value of the variation in the wavelength of the surface hybrid plasmonic resonance of the optical biosensor with the variation of the concentration of an estradiol solution in contact with said biosensor;

[0040] • Figure 9: bar chart of the specificity / selectivity of the invention's ultrasensitive biosensor compared to other analytes / substances such as bovine albumin (BSA) and dihydrotestosterone (DHT), tested in much higher concentration ranges of the specific analyte (estradiol).

[0041] Definitions

[0042] • In this document, the terms "approximately" or "about" as used herein when referring to a measurable value such as a quantity, a time duration, and the like, are intended to encompass variations of ±20%, ±10%, ±5%, ±1 %, or ±0.1 % from the specified value, where such variations are appropriate to perform the methods described.

[0043] • In this document, the terms, "comprises," "includes," "has," "having," "contains," "containing," "characterized by," or any other variation of these terms, are intended to cover a non-exclusive inclusion, subject to any limitations expressly stated. For instance, a composition, mixture, process, or method that comprises a list of elements is not necessarily limited to these elements but may comprise other elements not expressly listed or inherent in that composition, mixture, process, or method.

[0044] • In this document, in all embodiments of the present invention, the "upper face" of the optical sensor of the invention means the face that comprises the receptor covalently immobilized on the metal surface, on the other hand, the "lower face" of the optical sensor means the face that comprises the surface of the substrate 1000 opposite to that in direct contact with the first base layer 100, i.e. which comprises the lower surface 1001 .

[0045] • In this document, the term "nano-urchin / nano-urchins" refers to a three-dimensional anisotropic morphology, consisting of a central core adhering to (or in contact with) the plane or surface 240, from which nanometric protrusions (tips) oriented in space in multiple directions branch off. The morphology of nano-urchins on the plane or surface can be generically described as a three-dimensional anisotropic distribution of nanometric tips oriented radially or pseudo-radially with respect to the center of the particle but confined to the half-space outside the plane or support surface, with truncation of the spherical symmetry in the area of contact with the plane.

[0046] • In this document, the terms "inter-structure" and "intra-structure" are intended to comprise both pollen-based and urchin-based structures.

[0047] • In this document, the term "intra-pollen period" indicates the distance between two consecutive nanotips of the same pollen grain, wherein these nanotips are nanostructures that externally cover the surface of the pollen grains.

[0048] • In this document, the term "inter-pollen period" indicates the distance between two nanotips of two consecutive pollen units immobilized on the surface of the substrate.

[0049] • Similarly in this document, the term "intra-urchin period" indicates the distance between two consecutive nano-tips of the same urchin granule, wherein these nano-tips are nanostructures that externally cover the surface of the urchin granules.

[0050] • Similarly in this document, the term "inter-urchin period" indicates the distance between two nano-tips of two consecutive urchin units immobilized on the surface of the substrate.

[0051] • In this document, the term "sensor system" means an instrumental configuration for the detection of an analyte comprising at least one sensor chip according to the invention, at least one reference chip, at least one light source and at least one detection device (or detector).

[0052] • In this document, the terms "sensitive structure 200", "sensitive layer 200" and "sensitive zone 200" are to be considered synonyms and comprise periodic or pseudo-periodic nanostructures. In the present invention, the arrangement of nano-urchins and pollen grains on the 240 plane is, for instance, a random arrangement.

[0053] • An explanation of what is meant by the terms "periodic " and "pseudo-periodic" structure is given below. In hybrid plasmonic phenomena, metallic nanostructures hundreds of nanometers long couple with other metallic nanostructures, also hundreds of nanometers long. In hybrid plasmonic phenomena, the term "periodic" nanostructures is used to indicate that a nanometer structure (e.g. a line), typically hundreds of nanometers long, repeats periodically along a plane, with a period always of the order of tens or hundreds of nanometers. The term "pseudo-periodic" nanostructures is used to indicate that nanostructures behave like periodic ones, in terms of mutual coupling, since hybrid plasmonic phenomena are excited, in a similar way to periodic nanostructures, even if their performance is not optimized as in the periodic case. Thus, in terms of nanostructures, the sunflower pollen-based system can be considered a "pseudo-periodic" nanostructure, similar to those based on electron beam lithography studied in [ARCADIO F. et al: "Biochemical sensitive exploiting plasmonic sensors based on gold nanogratings and polymer optical fibers" PHOTONICS RESEARCH, vol. 9, no. 7 (2021 -07-01 ), XP093254963, ISSN: 2327-9125], wherein several "pseudoperiodic" nanostructures are simulated, manufactured, tested and compared by changing the width of the lines with a fixed spacing between them and, for a fixed width of the lines, by changing the spacing between the lines.

[0054] • In this document, the term "dielectric" indicates the solution, mixture or fluid (such as air, aeriforms, aerosols, aqueous solutions), also referred to as "bulk" or medium, which can optionally contain the analyte to be analyzed, with which both the sensor chip and the reference chip are in contact or are immersed; in particular, it is the medium placed in contact with the metal nanofilm functionalized with the receptor of the upper face of the chip sensor and with metallic reference chip nanofilm.

[0055] • In this document, the terms "sensor chip", "sensor", and "optical sensor" are used synonymously.

[0056] • In this document, the term "biosensor" is used to refer to the optical sensor of the invention wherein the layer of receptor 400 (not shown) comprises a bioreceptor, e.g. an antibody.

[0057] Detailed description

[0058] The present invention refers to an optical sensor for the specific and selective detection and quantification of one or more analytes within a sample under investigation. The optical sensor according to the present invention is a device comprising a plurality of overlapping layers, arranged in an orderly and functional manner for the purpose, and which on the surface presents a periodic or pseudo-periodic (semi-periodic) nanometric structure.

[0059] According to the present invention, the optical sensor, by externally exposing a nanostructured surface, is able to generate surface hybrid plasmonic resonances (e.g., due to a combination of SPR and LSPR) induced by an appropriate light stimulation, which can be recorded by an appropriate detection device.

[0060] The presence of a specific analyte interacting with receptors located on the upper face of the optical sensor of the invention is able to induce a variation (shift) of the wavelength (A) of hybrid plasmonic resonance; this variation of A may be related to the presence / quantity of the analyte of interest.

[0061] The periodic or pseudo-periodic nanometric structure that on the surface covers the upper face of the optical sensor (or sensor chip) according to the present invention can be natural or synthetic.

[0062] In the preferred embodiments of the present invention, a pseudoperiodic structure with tips with a distribution of the tips in space in the form of a urchin has been used, such as a structure of natural origin from pollen, preferably sunflower pollen as, for instance, the structures illustrated in Figures 1 a to 3c.

[0063] Without having to resort to lithographic techniques or complex manufacturing processes, it is possible to resort to the implementation of natural systems such as pollen within the optical sensor(s) of the invention. This pollen, suitably treated and coated with one or more nanometer-thick metal layers, is able to generate the aforementioned hybrid plasmonic phenomena due to a superficial pseudo-periodic nanometric structure.

[0064] With reference to the figures, an optical sensor based on a periodic or pseudo-periodic structure such as that of nano-urchins or sunflower pollen is shown, to detect the presence and / or concentration of an analyte in a matrix, comprising gaseous matrix, downstream of its functionalization with a natural or synthetic receptor, preferably specific to the type of analyte sought.

[0065] In this document, for the sake of simplicity, reference will be made to an illustrated system of XYZ Cartesian axes wherein the longitudinal X axis, the transverse Y axis and the vertical Z axis are respectively the longitudinal development axis, the width development axis and the height development axis of the optical sensor according to the present invention, as illustrated in Figure 1 e.

[0066] Further, the terms "top", "bottom" and "side" will refer to the relative position with respect to the invention ultrasensitive optical sensor configurations illustrated in Figure 1 e.

[0067] As shown in Figure 1 e, the ultrasensitive optical sensor according to the invention comprises in the lower part a guiding and transparent substrate 1000 which has a first surface or lower surface 1001 and a second surface or upper surface 1002 opposite each other and parallel with respect to the XY plane.

[0068] Said guiding and transparent substrate 1000 is made up of a material transparent to the electromagnetic radiation used for the detection of the analyte, wherein said electromagnetic radiation is comprised in the spectral region from ultraviolet to far infrared, preferably comprised in the visible spectrum (350 - 750 nm).

[0069] Said guiding and transparent substrate 1000 can have a thickness preferably between 500-1000 pm.

[0070] Said transparent material constituting the transparent and guiding substrate 1000 can be

[0071] • inorganic, such as but not limited to glass, silica, quartz, or

[0072] • organic, such as PMMA (Polymethyl Methacrylate) as a nonlimiting example.

[0073] The ultrasensitive optical sensor according to the present invention comprises a sensitive layer (structure, zone, region) 200 anchored to the upper surface 1002 of the transparent and guiding substrate 1000 by means of a base layer 100, which in turn is interposed between the surface 1002 of the substrate 1000 and the sensitive layer 200.

[0074] This base layer 100 has the function of binding the sensitive layer 200 to the transparent and guiding substrate 1000, and may consist of a material chosen as a non-limiting example between

[0075] • silanes such as aminosilanes, epoxysilanes;

[0076] • polymers, e.g. copolymers with succinimide functionality; which bind the sensitive layer 200 to the guiding and transparent substrate 1000, lying on a lying surface parallel to the XY plane.

[0077] The sensitive structure (or sensitive layer) 200 consists of a periodic or pseudo-periodic structure, of natural origin or of synthetic / artificial derivation (e.g., nano-urchins, i.e. synthetic structures that have nanometric protrusions on the surface) as illustrated in Figure 2a-2d and Figure 3a-3c.

[0078] The sensitive layer 200 is not obtained by lithographic techniques in any embodiment of the present invention.

[0079] Preferably, the sensible structure is a periodic or pseudo-periodic structure of natural origin.

[0080] Even more preferably, the periodic or pseudo-periodic natural structure is that of pollen, such as sunflower pollen (Figure 1 a), although pollen from a large class of plants can be implemented.

[0081] The nano-urchins, and preferably the pollen grains implemented in the preferred embodiment of the present invention, have on the external surface a periodic or pseudo-periodic pattern, (Figure 2b), normally pseudo-periodic, of nanostructures (e.g. nanotips) having a size of about one hundred nanometers, separated from each other by an interspace (intra-pollen period) of the order of magnitude of the wavelength of a visible radiation.

[0082] In more detail, • the inter-pollen period can be controlled by maintaining the same concentration of the pollen solution but varying the density of the chemical groups on base layer 100, i.e. the number of chemical groups capable of binding to pollen per unit area of base layer 100;

[0083] • the inter-pollen period can be controlled by changing the concentration of the pollen solution applied to substrate 1000 by means of base layer 100 (i.e., by the number of pollen grains immobilized on the substrate per unit area);

[0084] • the intra-pollen period is a function of the variety of pollen used or of similar natural or synthetic nanostructures used (e.g., nanourchins).

[0085] Preferably, the intra-pollen period varies between 100 nm and 50 pm while the inter-pollen period varies between 100 nm and 500 pm.

[0086] As for nano-urchins,

[0087] • similarly to pollen, the inter-urchin period can also be controlled by maintaining the same concentration and varying the density of the chemical groups on base layer 100;

[0088] • similarly to pollen, the inter-urchin period can be controlled by changing the concentration of the nano-urchin solution applied to substrate 1000 by means of base layer 100 (i.e., by the number of nano-urchins immobilized on the substrate per unit area);

[0089] • the intra-urchin period is a function of the size of the synthesized particle (e.g. from 50 to 200 nm).

[0090] The pollen, once a chemical process known [1 ,2] for the opening of the structure (Figure 1 b) and the formation / exposure of free surface carboxyl groups necessary for the formation of a chemical bond has been implemented, is chemically immobilized on the guiding and transparent substrate 1000 by means of the base layer 100, so as to expose the periodic or pseudo-periodic nanometric structural pattern to the outside. (Figure 1 c)

[0091] Preferably, when sensitive layer 200 consists of the natural periodic or pseudo-periodic structure of pollen, base layer 100 is made of compounds capable of binding the carboxyl groups present on the outer surface of the open pollen grains.

[0092] Nano-urchins are chemically synthesized inorganic-based structures with mainly metallic materials such as gold and silver or organic materials such as carbon. During synthesis, they can be externally modified with succinimidine (NHS) or maleimide groups in order to bind them, for instance, on the guiding and transparent substrate 1000 by means of the base layer 100 made of compounds capable of binding the -NHS or maleimide groups, for instance present on the external surface of nano-urchins.

[0093] The sensitive layer 200 is covered on the surface with a first metal layer 240.

[0094] In this case, the first metal layer 240 is in direct contact with the sensitive layer 200.

[0095] The first metal layer 240, in direct contact with the sensitive layer 200 and in the absence of further metal layers (which can be provided for in other embodiments of the invention) has a thickness between 10 nm and 70 nm.

[0096] The surface metallic coating (nanofilm) 240 deposited above the sensitive zone 200 may consist of but is not limited to

[0097] • a noble metal such as, by way of non-limiting example, gold, silver;

[0098] • a metal multilayer, such as, by way of example, silver / gold multilayer;

[0099] • a multilayer of metal oxides and metals such as, by way of nonlimiting example, zirconium / gold oxide, titanium / gold oxide;

[0100] • a metal alloy such as, by way of non-limiting example, gold / palladium.

[0101] In an optical sensor (sensor chip) embodiment, according to the invention, the first metal layer 240 is a gold film with a thickness of 10 nm to 70 nm. In another embodiment, a first intermediate layer 300 can be interposed between the first metal layer 240, preferably in gold, and the sensitive layer 200.

[0102] This first intermediate layer 300 can be made, by way of example but not limited to, in a material chosen from:

[0103] • chromium;

[0104] • titanium;

[0105] • titanium oxide (TiC>2);

[0106] • zirconium oxide (ZrC ); and combinations thereof, and it is preferably between 10 nm and 30 nm thick. The list of possible metallic materials to be used for the construction of this first intermediate layer 300 is to be intended as purely illustrative and not limiting towards the choice of additional and alternative metals or pairs of metals or metal oxides, to be implemented for reasons of instrumental performance (e.g. to improve optical sensitivity) and adhesion to the substrate.

[0107] According to the present embodiment wherein there are two metal layers, i.e. the first metal layer 240 and the first intermediate layer 300, the first intermediate layer 300 has a thickness between 10 nm and 30 nm while the first metal layer has a thickness between 10 and 70 nm.

[0108] The optical sensor according to the present invention comprises a layer of receptor 400 (not shown in the figures). More specifically, the layer of receptor 400 is chemically immobilized on the metal layer 240, comprising a suitable receptor of natural or synthetic origin, selectively chosen for the nature of the analyte sought.

[0109] This receptor has the function and is chosen in such a way as to interact selectively with a specific analyte present in the matrix to be examined.

[0110] The receptor can be but not limited to an antibody or a fragment thereof, a peptide, a nucleotide receptor such as the aptamer, a DNA / RNA probe or other types of natural or synthetic receptors. The receptor can further be a chemical receptor such as a polymer film on which is printed the shape of the target molecule or a mixture of target molecules when a class of substances is to be measured via the technology known as Molecularly Imprinted Polymer (MIP).

[0111] The immobilization of the receptor on the surface of the metal layer can take place directly using, by way of example, the chemistry of thiols (thiol groups have a high chemical affinity for noble metals such as gold and silver) or by using intermediate layers based on self-assembled monolayer (SAMs) that offer a wide variety of plausible chemical groupings (e.g., amines, carboxyls, epoxies). In any case, the immobilization methods are known to the expert in the field and can be chosen / adapted in relation to the nature of the receptor of interest to be anchored to the surface of the metal layer 240.

[0112] In an embodiment of the present invention, the receptor anchored to the metal layer 240 is a specific receptor of estradiol (ER, code: ab82606, manufactured by Abeam, Cambridge, UK). By using a lipoic acid layer to coat the metal surface 240 of the biosensor, the ER receptor can be immobilized above the lipoic acid layer with the coupling agent carbodiimide [3].

[0113] In a particularly preferred embodiment of the invention, the optical sensor according to the invention comprises:

[0114] • a transparent and guiding glass substrate 1000 with a thickness of 1000 pm;

[0115] • a base layer 100 in aminosilane having a density of amino groups of about 1014per cm2of transparent and guiding substrate 1000 adhered to the upper surface 1002 of said substrate 1000;

[0116] • a sensitive layer 200 in sunflower pollen (opened and prepared according to a process of known technique [1 ,2]) with a density of about 104pollen / cm2, anchored to substrate 1000 by means of base layer 100; • a metal layer 240 in gold with a thickness of 45 nm and deposited on top of the sensitive layer 200 by means of the "sputter coating" technique,

[0117] • a layer of receptor 400 comprising an estradiol receptor (ER) immobilized on metal layer 240 by means of lipoic acid and carbodiimide.

[0118] Therefore, the object of the present invention is an optical sensor (or sensor chip) made with nanostructured systems, preferably natural such as sunflower pollen, or alternatively synthetic, such as nano-urchins, which on the surface present a periodicity or pseudo-periodicity of nanostructures capable of generating hybrid plasmonic phenomena (by excitation from an incident radiation) once covered with metallic nanofilms (e.g., with a gold coating).

[0119] The sensor of the invention differs from known art not only for the presence of nanostructures with tips such as nano-urchins and pollen grains, but also for the way the signal is read, which in known art is carried out by measuring the amplitude / intensity of the plasmon response (or combined with PEF / SERS). Unlike in the sensor of the present invention, the key feature is the determination of the wavelength shift of the plasmonic peak based on hybrid modes (spectral variation), which provides a detection criterion that is not only different, but also more precise and reliable. In fact, the sensor of the present invention is characterized by the fact that the detection of the analyte is carried out by the determination of a wavelength shift of the maximum plasmonic resonance, as a consequence of the interaction between the receptor layer and the target analyte.

[0120] Therefore, the optical sensor of the invention is a sensor for the detection of an analyte in a sample by means of hybrid plasmonic phenomena, said sensor comprising: a transparent and guiding substrate 1000 having a lower surface 1001 and an upper surface 1002 parallel and opposite each other, said substrate being transparent to the electromagnetic radiation used for the detection of the analyte, said electromagnetic radiation being comprised in the spectral region from ultraviolet to far infrared;

[0121] • a base layer 100 in contact with the upper face 1002 of said substrate 1000;

[0122] • a sensitive layer 200 chemically immobilized on the guiding and transparent substrate 1000 by means of base layer 100;

[0123] • a first metal layer 240 to cover the upper surface of said sensitive layer 200,

[0124] • a layer of receptor 400 chemically bound to the upper surface of said first metal layer 240; wherein: the sensitive layer 200 comprises a plurality of nanostructured systems, chosen from nano-urchins and pollen grains, capable of generating hybrid plasmonic phenomena in combination with the metal layer 240 by interaction with electromagnetic radiation, and wherein: the layer of receptor 400 comprises a receptor chosen from: an antibody or a fragment thereof, a peptide, a nucleotide receptor such as the aptamer, a DNA / RNA probe, a chemical receptor such as a polymer film on which the shape of the target molecule is printed or a mixture of target molecules; this optical sensor being configured to allow the detection of the analyte by determining the wavelength shift of the maximum plasmonic resonance.

[0125] This determination provides a more precise and sensitive measurement than detection based on plasmon band width alone.

[0126] The sensor is also characterized by the fact that it comprises a spectral analysis module configured to detect the displacement of the plasmonic peak in wavelength, wherein this displacement represents the discriminating parameter for the presence and / or concentration of the analyte, with greater precision and sensitivity than the measurement of variations in intensity or amplitude of the plasmonic band.

[0127] Nanostructuring and hybrid plasmonic resonance.

[0128] The sensitive layer 200, being made up of a periodic or pseudoperiodic structure chemically linked to the base layer 100, on the surface presents a spatial repetition of periodic or pseudo-periodic nanostructures (i.e., plurality of nanotips). The particular nanometric geometry presented by this system is responsible for the interaction with electromagnetic radiation and the generation of hybrid plasmonic phenomena at the metalreceptor interface. In fact, the sensitive layer 200 and the first metal layer 240 that covers it have shape, optical properties, dimensions and period (inter-structure and intra-structure) such that, when the optical signal (e.g., electromagnetic radiation in the visible spectrum) impinges on the first metal layer 240, a hybrid plasmonic phenomenon (due to the mutual interaction between SPR and LSPR) is excited at the interface between the upper face of the optical sensor and the dielectric (solution / solvent / mixture / gaseous fluid, hereinafter referred to as "bulk") wherein it is immersed or with which it is in contact, at a given wavelength.

[0129] In particular, the hybrid plasmon is excited at a specific wavelength due to the pseudo-periodicity of the nanostructures, therefore by the mutual coupling between the nanotips in the case of pollen or nanourchins, which act as coupled plasmonic nano-antennae. In the case of sunflower pollen, for instance, this occurs both within the same pollen (intra-pollen) and on several contiguous pollen units (inter-pollen).

[0130] The excitation wavelength of the plasmon is a function of numerous parameters, comprising the shape of the nanostructure, the size, the periodicity (intra-structure period and inter-structure period), the metallic material, the dielectric nature of the bulk and, above all, the possible presence of analytes capable of interacting with the surface receptor of the optical sensor of the present invention.

[0131] In fact, when the sensor chip of the invention, functionalized with a receptor (natural or synthetic) specific for an analyte, is put in contact with a fluid such as a liquid matrix, a solution, air, aeriform, or an aerosol containing said analyte, the analyte binds to the receptor and varies its optical properties, i.e. the refractive index at the interface between the sensitive face of the optical sensor chip and the bulk, changing plasmonic resonance conditions. The detection method is based on the fact that the variation of optical properties due to binding to the analyte results in the shift of the plasmonic resonance wavelength. This spectral shift is related to the concentration value of the analyte in the solution. For instance, in the linear part of the dose-response curve, the resonance shift is proportional to the change in analyte concentration relative to blank / medium (resonance value in the medium in the absence of analyte).

[0132] The pseudo-periodicity of nanostructures (natural or synthetic), such as those of pollen or nano-urchins, lead to the excitation of hybrid plasmonic modes that are typical of periodic or pseudo-periodic structures obtained through lithographic techniques aimed at creating regular structures of a few hundred nanometers. Hybrid plasmons compared to SPR and LSPR phenomena considered individually allow an improvement in the performance of the optical sensor such as to reach, as in the case of proteins, lower detection limits.

[0133] Sensor properties.

[0134] In particular, as highlighted in the Examples section (Example 2) of this document, the optical sensitivity of the sensor chip (optical sensor) has been evaluated according to the present invention defined as

[0135] 5A / bn wherein A is the wavelength of the resonance, n is the refractive index of the bulk placed in contact with the upper face of the optical sensor and 5 the differential. In the absence of a layer of the 400 receptor, it has been ascertained that changing the bulk in contact with the metal surface 240 changes the wavelength at which the hybrid plasmon is induced. In fact, the plasmonic phenomenon is strictly dependent on the refractive index at the interface between the metal medium on which it originates and the bulk wherein the sensor chip is immersed (or with which it is in contact).

[0136] The binding sensitivity of the optical sensor was further evaluated according to the present invention. Surprisingly, the optical sensor of the invention has shown to possess instrumental sensitivity in the order of the attomolar concentration. As will be appropriately explained in the Examples section (Example 3) of this document, the experimental evidence supporting this non-inferable sensitivity in analyte detection was obtained by evaluating the variation of the wavelength of the hybrid plasmon on the ultrasensitive optical sensor functionalized with the estradiol receptor (ER) as the concentration of the estradiol solution itself changes.

[0137] The measurement range of the substance of interest (analyte) can be modified / customized by acting on various parameters of the sensor system proposed in this description, comprising the possibility of changing the performance / sensitivity of the optical sensor by acting on the concentration / density of the pollen anchored / immobilized on the sensitive surface, on the type of periodic or pseudo-periodic structure used (natural or synthetic), with the possibility of using different nanostructures, on the number and type of metal nanofilms used to obtain the hybrid plasmonic phenomenon, on the type of natural or synthetic receptor used, with the possibility of using receptors with different efficiencies (wherein efficiency means the ability of the receptor to change its refractive index when binding to the analyte occurs).

[0138] Another object of the present invention is a system of sensors, i.e. a plurality of ultra-sensitive optical sensors comprising periodic or pseudoperiodic structures, preferably of natural origin, having a structure like that of Figure 1 e, solidly placed side by side.

[0139] This sensor system comprises a plurality of optical sensors arranged on the same geometric plane and according to any lattice geometry (diagram) (Figure 4a), wherein each optical sensor of the plurality of optical sensors of said sensor system has a layer of receptor 400 specific for the detection of a different analyte.

[0140] Another object of the present invention is an optical multisensor (Figure 4b). This optical multisensor has the same multilayer structure as the sensor chip of the invention except for the layer of receptor 400. More specifically, the surface of the metal layer 240 of this optical multisensor is not uniformly coated by a single layer of receptor 400 but can be covered by a plurality of layers of receptor 400, different from each other, to form distinct surface regions, according to any geometry, each specific for a given analyte.

[0141] The sensor system according to the invention and the optical multisensor according to the invention are referred to in this document more generally as array (or sensor array).

[0142] Preferably, said sensor array is capable of detecting a large class of analytes at the same time.

[0143] A further object of the present invention is an apparatus also called a sensor system for the qualitative and / or quantitative detection of an analyte in a medium such as a liquid / gaseous solution or air, or an aerosol, comprising

[0144] • at least one optical ultrasensitive sensor (also called sensor chip) according to the invention, or an array of sensors;

[0145] • at least one reference chip;

[0146] • at least one source of electromagnetic radiation in the ultraviolet to far-infrared spectral region;

[0147] • at least one detection device (or detector);

[0148] • at least one sample holder (holder); wherein, the at least one source of electromagnetic radiation can be, by way of example but not limited to, an LED or a light source that emits in the visible range; • the at least one detector may be chosen by way of example but not limited to by the group comprising a spectrometer, a spectrophotometer or a photodiode; and

[0149] • the at least one reference chip comprises the same sequence of layers as the transparent and guiding substrate 1000 and layer 240 of the sensor chip of the invention even if it is deprived of the sensitive layer 200 and the receptor layer 400, wherein said at least one reference chip is placed on the same base plane XY as the sensor chip / sensor array (the metal surfaces of the sensor chip / sensor array and the chip lie in the same XY plane) and is used for normalization of the signal(s) obtained from the sensor chip / sensor array (e.g. for normalization of transmission spectra).

[0150] In a preferred form of sensor system implementation described in Figure 5a, the at least one source of electromagnetic radiation is a white light lamp (i.e., emitting in the visible spectrum 350-750 nm) and the at least one detection device is a spectrophotometer. In the present embodiment of the sensor system in Figure 5a, two spectrophotometers are provided, one for capturing the signal transmitted by the sensor chip, the other for capturing the signal transmitted by the reference chip.

[0151] In an embodiment of the sensor system (Figure 5b), the at least one source of electromagnetic radiation and the at least one detection device are respectively implemented within at least a first and a second optoelectronic board distinct from each other, lying on two planes parallel to the XY base plane and located on opposite sides to at least one optical sensor / sensor chip / optical biosensor. Each may comprise electronic or photonic systems aimed at improving the signal-to-noise ratio of the sensor system. By way of example, the optoelectronic board, on the source side, can comprise integrated optoelectronic elements for intensity or phase modulation, wavelength tuning and other techniques well known to the expert in the field of telecommunications to make the sensor system performing. The at least one first optoelectronic board comprising at least one source of electromagnetic radiation, comprised in the ultraviolet to far- infrared spectral region, is oriented towards the upper face of the at least one optical sensor / biosensor or sensor array, and the at least one second optoelectronic board comprising at least one sensitive device is oriented towards the bottom surface 1002 of the substrate of the at least one optical sensor or sensor array.

[0152] In another embodiment, configurations based on the excitation of the hybrid plasmonic phenomenon by evanescent field can be used, i.e. using substrate 1000 as the "core" of an optical waveguide and not as a transparent substrate as in the case of plasmonic nanostructures obtained by lithographic techniques [4,5]. In this case, the light propagating in the waveguide in question, which by structure can be considered multimodal, excites hybrid plasmonic phenomena using the evanescent field.

[0153] In other embodiments wherein the ultra-sensitive optical sensor chip of the invention and the reference chip for signal normalization are present at the same time, the sensor system can provide any combination of illumination technique and detection technology at the input and output of the sensitive structure (sensor chip) and the reference chip.

[0154] The illumination of the ultra-sensitive optical sensor and the reference chip can be obtained without the aid of input fibers, but with a single light source and a single input lens. Detection, on the other hand, takes place for both structures by means of two respective output fibers connected to the respective detectors or to the respective inputs of the same detector, allowing the collection of the light transmitted through the two chips and conveying it to the detector itself.

[0155] In another embodiment, the sensor system consists of a configuration based on LED-photodiode coupling. The light source emits an optical signal in a predetermined wavelength range, for instance in the red, green or blue wavelength range, and the concentration of the analyte is then determined on the basis of the change in intensity of the detected optical signal. In particular, in the case of the presence of the analyte, there is a variation in signal intensity with respect to the intensity of the reference optical signal detected in the absence of the analyte itself. The variation in the intensity of the optical signal is related to the change in the plasmonic resonance conditions due to the variation of the refractive index of the receptor layer that binds to the analyte, and, therefore, to the concentration of the analyte to be detected.

[0156] In another embodiment, the optical signal generated by the light source is white light (for instance a range between 350-750 nm); in this case, the concentration of the analyte is determined on the basis of the spectral shift of the plasmonic resonance detected by a detection device, e.g. a spectrophotometer, with respect to the reference optical signal detected in the absence of the analyte itself.

[0157] In the event that the instrumental setup involves a reference chip next to the ultrasensitive optical sensor of the invention (such as the one shown in Figure 1 e) (identical to the ultrasensitive optical sensor but deprived of the sensitive layer 200 and the receptor layer 400), the optical signal output from the reference chip is used to normalize the optical signal output from the sensor chip.

[0158] PRODUCTION METHOD

[0159] A further object of the present invention is the method (process) for the realization of the ultra-sensitive optical sensor of the invention. The method according to the present invention can be realized through the procedure described below or with slight modifications of it, which in any case are within the knowledge of the expert in the field.

[0160] In particular, the realization of a single ultrasensitive optical sensor according to the present invention will be described, comprising a sensitive structure 200, and a reference chip, since this description can be extended to the realization of the sensor system (e.g., array) by any expert in the field (Figure 4).

[0161] The method for realizing the ultra-sensitive optical sensor according to the present invention comprises the steps of • preparing the substrate 1000;

[0162] • immobilizing the sensitive layer 200 on the upper surface 1002 of substrate 1000 with techniques known to the expert in the field, preferably consisting of pollen [1 ,2] or nano-urchins [3] by modifying substrate 1000 with a suitable base layer 100, appropriately chosen according to the chemical nature of substrate 1000;

[0163] • optionally depositing the first intermediate layer 300, using techniques such as e.g. "sputter coating", evaporation or alternative nanometer film deposition techniques known to the expert in the field;

[0164] • depositing the first metal layer 240 in direct contact with the sensitive surface 200 by means of the sputtering technique or by evaporation, or in case the optional first intermediate layer 300 has been deposited, in direct contact with said first intermediate layer 300;

[0165] • chemically binding the receptor of the receptor layer 400 (or more receptors in the case of an optical multisensor in Figure 4b) to the first metal layer 240, using techniques known to the expert in the field.

[0166] Further details are described in an embodiment of the optical sensor manufacturing method of the invention given in the Examples section of this document (Example 1 ).

[0167] The method of making the reference chip is similar to that of the realization of the ultrasensitive optical sensor of the invention but excluding the immobilization phase of the sensitive layer 200 and the layer of the receptor 400. Any expert in the field, on the basis of knowledge of known technique, will not encounter any difficulty in carrying it out.

[0168] The procedure described above can be implemented in such a way as to obtain the array of sensors in Figure 4a, i.e. a plurality of optical sensors solidly side by side in the configuration of a system of sensors. METHOD OF USE

[0169] The procedure to be performed for the use of the sensor according to the invention for the determination of the presence / quantity of a specific analyte in a sample is described below. The method of use according to the present invention comprises the steps of:

[0170] • allocating the sensor chip and the reference chip in the sensor system having the sample holder connected to the optoelectronic devices, i.e. to at least one source and at least one detector;

[0171] • acquiring the output signals such as, for instance, the spectra in transmission when illuminated with a white light source from the sensor chip and the reference chip, obtained by placing the blank, i.e. the medium (bulk) without analyte, in contact with them;

[0172] • normalizing the output signal from the sensor chip to the output signal from the reference chip, such as for each wavelength of the obtained spectrum;

[0173] • reading the normalized signal, such as the resonance wavelength or the intensity of the transmitted radiation;

[0174] • immersing the sensor chip and the reference chip in the sample to be analyzed, whether liquid or gaseous, or in an aerosol state;

[0175] • incubating the medium containing the analyte of interest for about 5 minutes so that the analyte remains bound to the specific receptor layer of the sensor chip;

[0176] • washing, if required by the medium being analyzed, with for instance phosphate buffer (PBS), to remove any non-specific bonds between the surface and possible interferents;

[0177] • acquiring the output signals such as the spectra in transmission when illuminating with a white light source from the sensor chip and the reference chip, obtained by putting the blank in contact with them, i.e. solution without analyte; • normalizing the output signal from the sensor chip to the output signal from the reference chip, such as for each wavelength of the obtained spectrum;

[0178] • reading the normalized signal, such as the resonance wavelength or the intensity of the transmitted radiation;

[0179] • detecting the variation of the signal with respect to the blank, i.e. the signal acquired, for instance in PBS, before incubation with the medium containing the analyte. For instance, the change in the signal can be the shift in the resonance wavelength, which in the linear part of the dose-response curve (usually a sigmoid) is proportional to the change in the concentration of the substance of interest (analyte).

[0180] An advantage of the optical sensor of the invention is the high sensitivity to perturbations of boundary conditions.

[0181] A further advantage of the invention's ultrasensitive optical sensor based on natural structures such as pollen lies in the fact that a resonance phenomenon is triggered even when air is present as a surrounding medium (Figure 6a), making it possible to use it also in matrices such as air or aeriform, for the detection of hazardous substances, or aerosols, for the detection of bacteria and viruses, without limiting their use to aqueous solutions only.

[0182] The ultra-sensitive optical sensor based on natural nanostructures, such as those of pollen or other similar structures, comprising artificial ones such as nano-urchins, according to the present invention, is simple and easy to couple with a light source and an optical detection device, which can be arranged on opposite sides of the optical sensor in a simple way and in a small space.

[0183] Advantageously, the interrogation technique is based on the measurement of the variation of the resonance wavelength (wavelength coding) so it is immune to any variation in the intensity of the signals due to external factors such as fluctuations in the power supply, vibrations, bending and / or manipulation of the optical fibers used to connect the source to the sensor and the output of the sensor to the spectrometer. It is therefore extremely more robust than similar configurations which, although based on localized plasmonic resonance (LSPR), instead detect the presence of the analyte by measuring the changes in absorbance of the sample following the interaction of the molecular recognition element with the analyte.

[0184] Advantageously, the measurement range of the substance of interest (analyte) can be modified / customized by acting on various parameters of the sensor system proposed in this description, comprising the possibility of changing the performance / sensitivity of the sensor chip by acting on the concentration / density of the nano-urchins or pollen anchored on the sensitive surface, on the type of nano-urchins or pollen used (natural or synthetic), with the possibility of using different nanostructures, on the number and type of metal nanofilms used to obtain the hybrid plasmonic phenomenon, on the type of natural or synthetic receptor used, with the possibility of using receptors with different efficiencies (wherein efficiency means the ability of the receptor to change its refractive index when binding to the analyte occurs). Finally, it is possible to modulate the sensitivity of the sensor system by acting on the optoelectronic setup.

[0185] Advantageously, ultra-sensitive optical sensors based on natural structures such as pollen, or artificial ones such as nano-urchins, according to the present invention, are very versatile and can be combined with each other according to any scheme in such a way as to realize the sensor system (e.g., sensor array) described above. This sensor array may be able to simultaneously detect the presence and / or concentration of several different analytes, exploiting the specificity / selectivity (induced by the receptor layer) of each individual sensor.

[0186] The sensor system according to the present invention is advantageously simple to realize, not requiring multiple depositions of silicone or epoxy materials hundreds of microns thick, nor complex systems of lenses and mirrors for the coupling of light both in and out of the optical sensor. Advantageously, the process for the realization of the ultrasensitive optical sensor based on natural nanostructures such as pollen nanostructures, or artificial ones such as nano-urchins, according to the present invention, is suitable for use for large-scale production, as it can be automated and parallelized.

[0187] Advantageously, compared to processes of known art that require complex and economically expensive techniques (e.g., lithographic techniques such as electronic or optical lithography), the method for the realization of the optical sensor according to the invention is simpler, less expensive and easily scalable.

[0188] Advantageously, the invention is able to implement ready-to-use periodic or semi-periodic nanostructures in the optical sensors of the present invention, made available by nature, rather than having to resort to the use of artificially derived periodic systems such as those obtained for instance with lithographic techniques.

[0189] However, there is no constraint on the implementation in the ultrasensitive optical sensor of the invention of synthetic nanostructures such as, but not limited to, nano-urchins (having a shape similar to that of the natural nanostructures covered by the present invention).

[0190] Advantageously, by means of the phenomenon of hybrid surface plasmonic resonance generated by the interaction between SPR and LSPR plasmonic phenomena, the optical sensor according to the present invention is able to exhibit a limit of detection (LOD) in the order of concentration of 0.3 attomolar which is lower than that obtainable with structures made with electron lithography [4,5] and previously implemented equal to 0.7 attomolar.

[0191] Furthermore, the ultrasensitive optical sensor / biosensor according to the invention is advantageously selective / specific towards the analyte of interest. As reported in more detail in the Examples section (Example 5) of this document, the invention sensor is able to discern the analyte of interest from the possible presence of potential interferents. This advantageous feature of the present invention is a function of the specificity of the receptor / bioreceptor itself anchored to the optical sensor of the invention.

[0192] Finally, it is clear that the ultra-sensitive optical sensor based on natural nanostructures such as those of pollen or even artificial ones such as those of nano-urchins, alone or in a system of sensors, the sensor system and the process of making the optical sensor conceived in this way are susceptible to numerous modifications and variations, all of which fall within the scope of the invention; in addition, all details are replaceable by technically equivalent elements. In practice, the materials used, as well as the dimensions, can be any depending on technical needs.

[0193] A further object of the present invention is a portable sensor system (or portable kit) for the field measurement of one or more analytes comprising:

[0194] • the ultra-sensitive optical sensor according to the present invention, or a sensor array;

[0195] • the reference chip

[0196] • a sample holder;

[0197] • at least one light source, preferably comprised in an optoelectronic board;

[0198] • at least one detection device, preferably comprised in an optoelectronic board;

[0199] • instructions for use, optionally accessible remotely through an electronic medium.

[0200] Optionally, the portable sensor system can comprise

[0201] • computerized means for analysing the data provided by the sensor comprising at least one processor, one storage memory and means for displaying data.

[0202] USE OF THE SENSOR The ultrasensitive optical sensor according to the invention functionalized with an appropriate receptor (chosen according to the nature of the analyte sought or measured) can be used for the selective detection of the presence and / or for the quantification / measurement / analysis of the concentration / quantity of said analyte in different matrices in numerous fields of application comprising, by way of example, the diagnosis of tumor markers, the analysis of various emerging environmental pollutants (in rivers, lakes, seas, farmland, wooded areas), bioterrorism or biodefense, the detection of viruses and bacteria, pandemic emergencies, personalized medicine, Point-of-care tests for different analyses or pre-analyses, tests on rare diseases, measurement of explosives.

[0203] EXAMPLES

[0204] The embodiments exemplary and not limiting the scope of the present invention reported below have been obtained by using as raw materials:

[0205] Sunflower pollen was purchased from Greer Labs (Lenoir, NC, USA). 1 -ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC, 22980) and N-hydroxysulfosuccinimide (Sulfo-NHS, 24510) were purchased by Fisher Scientific Italia (Milan, Italy). [3-estradiol (E8875, PM 272.38), ct-lipoic acid, ethanolamine, dihydrotestosterone (DHT, PM 290.44), bovine serum albumin (BSA) were purchased from Sigma Aldrich (Milan, Italy). The ERa receptor (ab82606) was purchased from Abeam (Cambridge, UK).

[0206] The aminosilane-coated substrates called "Nexterion A+" are purchased from SCHOTT Technical Glass Solutions GmbH (Jena, Germany).

[0207] The light source (model HL-2000LL) and spectrophotometers (model FLAME-S-VIS-NIR-ES) were purchased from Ocean Insight (Orlando, FL, USA).

[0208] The launch / read POFs, with a total diameter of 1 mm, were purchased from Edmund Optics (Barrington, NJ, USA). Example 1

[0209] Exemplary embodiment of the sensor of the invention.

[0210] A procedure for making the invention's ultrasensitive optical sensor comprises the following steps:

[0211] • pollen preparation by microgel formation according to the known art procedure [1 ,2] which consists of an initial removal of the cytoplasm by placing 1 g of defatted pollen in a round-bottomed flask with 10 mL of 10% (w / v) aqueous solution of KOH. The suspension is then stirred for 2 hours at 80°C under magnetic stirring. Then, the suspension is centrifuged at 3,500 rpm for 5 min. The supernatant is discarded, and the resulting pellet is coated with 20 mL of fresh 10% (w / v) KOH solution. The mixture is stirred at high speed for 2 minutes and then centrifuged at 3,500 rpm for 5 minutes, repeating this washing step four more times. The formation of the microgel is thus obtained by placing 10 mL of fresh 10% KOH solution (w / v) on the pellet and transferring the contents to the round-bottomed flask, repeating the heat treatment at 80°C for 6 h. Then the solution is centrifuged with the above procedure until the pH of the supernatant approaches neutral. The pellet is then kept at 4°C until use. At the end of this procedure, a carboxyl activated pollen shell is obtained.

[0212] • immobilization of pollen as prepared in the previous step on amine substrates covering the glass substrate 1000 through the chemistry of carbodiimides. 20 mg of pollen is dispersed in 0.5 mL of MES buffer (100 mM MES, 0.5 M NaCI, pH 6.0), and 52 mM of EDC and 138 mM of Sulfo-NHS are added to the solution which is mixed at 120 rpm in an orbital stirrer for 30 minutes. Then, the solution is centrifuged at 3,500 rpm for 5 minutes and the supernatant is replaced with phosphate buffer (phosphate buffer 10 mM, NaC1 138 mM, KCI 2.7 mM, pH 7.4). A drop of activated pollen solution is deposited on the surface and a 1 -hour incubation with orbital stirring is performed. • deposition of a 45 nm thick gold nanolayer by sputter coating technique;

[0213] • functionalization of the gold surface with the estradiol receptor (ER). To remove any hydrocarbon contaminants from the gold surface, an Argon plasma is applied for 2 minutes at 6.8 W. A 0.3 mM lipoic acid solution is applied to water overnight. After washing the surface in water, a solution of EDC / sNHS 10 / 10 mM in 50 mM MES pH 5.5 is applied for 30 minutes at 100 rpm in an orbital stirrer. After washing in MES buffer, 4 pg of estradiol receptor (ERa) is incubated in phosphate buffer for 2 hours and finally passivated with 1 mM ethanolamine in water for 30 minutes.

[0214] Example 2

[0215] Optical sensitivity of the sensor.

[0216] Figure 6a shows the spectra of normalized hybrid plasmonic resonance of an embodiment of the ultrasensitive optical sensor of the invention such as the one depicted in Figure 1 e comprising the transparent and guiding substrate 1000 in glass with a thickness of 1 mm and covered by the base layer 100 in aminosilanes, the sensitive surface 200 consisting of open sunflower pollen grains (according to known technique [1 ,2]) fixed on the substrate 1000 by means of the base layer 100, the metal layer 240 in gold with a thickness of 45 nm to coat (in direct contact) the sensitive layer 200 but without functionalization with the layer of the receptor 400 (natural or synthetic selective layer). The measurements were performed by placing the first metal layer 240 in contact with different "bulks" (liquid solutions of water and glycerine at different mixing ratios) having different refractive index (n). In the present example, 4 spectral recording measurements have been carried out with the following dielectrics: air (n=1 ) and 3 solutions having 3 different refractive indices listed below:

[0217] • Solution 1 : n = 1 .332

[0218] • Solution 2: n = 1 .343

[0219] • Solution 3: n = 1.355 The spectra recorded in Figure 6a clearly show how the refractive index at the metal-bulk interface varies (due to the change in the composition of the dielectric medium in contact with the metal layer 240) the excitation wavelength of the hybrid plasmon changes. In particular, the resonance wavelength decreases as the refractive index of the bulk increases. The transmission spectra obtained with the invention's ultrasensitive optical sensor were normalized with the spectra transmitted through the reference chip placed in contact with the same solutions.

[0220] Starting from the data contained in the graph of Figure 6a, a further graph (Figure 6b) has been developed that shows the absolute value of the variation of the resonance wavelength towards the refractive index of the external medium both for the ultrasensitive plasmonic optical sensor based on pseudo-periodic natural nanostructures, such as that of sunflower pollen, and a standard comparison sensor based on a nanolattice produced by electron beam lithography (EBL). [5]

[0221] Comparing the linear fitting curves obtained by the 2 sensors under examination, it can be seen that the optical sensor of the invention is more sensitive than the standard comparison sensor having an optical sensitivity value (6A / 6n), corresponding to the higher angular coefficient of the lines in Figure 6b:

[0222] 5A / 5n of the invention sensor = 773 nm / RIU

[0223] 5A / 5n of the standard sensor = 547 nm / RIU

[0224] Example 3

[0225] Binding sensitivity of the optical sensor.

[0226] The curves shown in Figures 7 and 8 refer to an embodiment of the ultrasensitive optical sensor of the invention such as the one depicted in Figure 1 e comprising the transparent and guiding substrate 1000 in glass with a thickness of 1 mm and covered by the base layer 100 in aminosilanes, the sensitive surface 200 consisting of open sunflower pollen granules (according to known technique [1 ,2]) fixed on the substrate 1000 by means of the base 100, the 45 nm thick gold metal layer 240 to coat (in direct contact) the sensitive layer 200 and further functionalized with a specific bioreceptor (receptor layer 400) for estradiol (estrogenic receptor, ER) according to the technique reported in the description and which involves the use of 1 -ethyl-3(3-dimethylaminopropyl) carbodiimide and ct-lipoic acid. The measurements were performed by placing liquid solutions at different concentrations of estradiol in contact with ER anchored to the optical sensor in order to obtain a dose-response curve and demonstrate that the binding sensitivity achieved by the ultrasensitive optical biosensor based on pseudo-periodic natural nanostructures such as those of pollen according to the present invention is of the order of attomolar.

[0227] The transmitted spectra obtained with the invention's ultrasensitive optical sensor were normalized with the transmitted spectra acquired by the reference chip for the same estradiol solutions.

[0228] In particular, Figure 7 shows a comparative graph of transmission spectra measured at wavelength variation on aqueous solutions of estradiol at different concentrations between 0.5 aM to 1000 aM.

[0229] As can be seen from the same figure, as the concentration of estradiol in solution changes, a shift in the transmission curve recorded by the optical sensor is observed, demonstrating how the instrument itself is sensitive to the variation in concentration of the analyte sought. Not only that, as the spectra in Figure 7 demonstrate, the instrument is sensitive to minimal variations in concentrations that even reach a tenth of the attomolar.

[0230] Figure 8 shows a dose-response curve (interpolated with a classical analyte-receptor interaction model, i.e. Langmuir model) obtained from the absolute value of the variation of the hybrid plasmonic resonance length of the invention sensor as a function of estradiol concentration.

[0231] Example 4

[0232] Configuring a sensor system in the presence of the optical sensor chip and the reference chip. Figure 5 schematically shows the case of a sensor system comprising the ultrasensitive optical sensor of the invention (as prepared in example 1 ) and the reference chip (i.e. an identical one but without pollen, i.e. without nanostructures) used for the normalization of the spectra. In this case, the sensor system has two multimode fibers (total diameter 1 mm) input to illuminate the ultra-sensitive sensor and the reference chip respectively, and two respective multimode output fibers to collect the light transmitted by both.

[0233] Example 5

[0234] Optical sensor selectivity of the invention.

[0235] The specificity / selectivity of the ultrasensitive optical sensor of the invention was further measured, having the thickness of the gold metal layer 240 equal to 45 nm, in direct contact with the sensitive surface 200, consisting in turn of open sunflower pollen granules (according to known technique) immobilized on substrate 1000, and functionalized on metal layer 240 with a bioreceptor specific for estradiol (estrogenic receptor, ER) according to the technique described in the description and which involves the use of lipoic acid and carbodiimide.

[0236] In particular, the selectivity of the biosensor was evaluated by comparing in a vertical bar graph (Figure 9) the variation of the wavelength of surface hybrid plasmonic resonance of the optical sensor of the present invention put in contact with 3 different solutions:

[0237] • an aqueous solution of 10 aM estradiol;

[0238] • an aqueous solution of bovine albumin (BSA) 1 f M; and

[0239] • an aqueous solution of dihydrotestosterone (DHT) 1 fM.

[0240] As can be seen from Figure 9, the optical sensor of the invention, by means of the intrinsic specificity of the estradiol bioreceptor (ER), showed a clear selectivity towards the analyte of interest, i.e. estradiol, despite the use of interferents at considerably higher concentrations (100 times more concentrated) than that of estradiol.

[0241] In conclusion, the device described in WO 2022 / 189802 is fundamentally different from that of the present invention in that it leverages LSPR only to enhance fluorescence excitation. Furthermore, it does not indicate a precise sensitivity but only an improvement compared to other fluorescence-based techniques such as ELISA.

[0242] The system described in ARCADIO et al. uses artificial nanostructures made by complex electron beam lithography techniques and has a sensitivity equal to about half that based on pollen. Therefore, the invention makes it possible to create a particularly reliable and extremely sensitive sensor, constituting a non-obvious improvement on the sensors of the known art.

[0243] References

[0244] [1] https: / / doi.Org / https: / / doi.org / 10.1016 / j.apmt.2020.100702;

[0245] [2] https: / / doi.Org / 10.1038 / s41467-020-15294-w;

[0246] [3] Bioconjugate Techniques 3rd Edition - July 25, 2013; Author: Greg T. Hermanson; Hardback ISBN: 9780123822390; eBook ISBN: 9780123822406;

[0247] [4] https: / / doi.Org / 10.3390 / nano11081961 .

[0248] [5] [https: / / doi.Org / 10.1364 / PRJ.424006].

Claims

CLAIMS1. An optical sensor for the detection of an analyte in a sample by localized plasmonic resonance, said sensor, comprising:• a transparent and guiding substrate 1000 having a lower surface 1001 and an upper surface 1002 parallel and opposite each other, said substrate being transparent to the electromagnetic radiation used for the detection of the analyte, said electromagnetic radiation being comprised in the spectral region from ultraviolet to far infrared;• a base layer 100 in contact with the upper face 1002 of said substrate 1000;• a sensitive layer 200 chemically immobilized on the guiding and transparent substrate 1000 by means of base layer 100;• a first metal layer 240 to cover the upper surface of said sensitive layer 200,• a layer of receptor 400 chemically bound to the upper surface of said first metal layer 240; wherein: the sensitive layer 200 comprises a plurality of nanostructured systems, chosen from nano-urchins and pollen grains, capable of generating, in combination with the metal layer 240 by interaction with electromagnetic radiation, surface hybrid plasmonic resonances, and wherein: the layer of receptor 400 comprises a receptor chosen from: an antibody or a fragment thereof, a peptide, a nucleotide receptor such as the aptamer, a DNA / RNA probe, a chemical receptor such as a polymer film whereon the shape of the target molecule or a mixture oftarget molecules is printed; said optical sensor being configured to allow the detection of an analyte by determining the wavelength shift of the maximum plasmonic resonance.

2. The optical sensor according to claim 1 wherein: the material the transparent and guiding substrate 1000 is made of is chosen from glass, quartz, silica, polymethylmethacrylate (PMMA), and is transparent to visible radiation in the range 350- 750 nm.

3. The optical sensor according to anyone of claims 1 -2 wherein: the material the base layer 100 is made of is chosen from• silanes, preferably chosen from aminosilanes, epoxysilanes;• polymers, preferably chosen from copolymers with succinimide functionality.

4. The optical sensor according to anyone of claims 1 -3 wherein: the material the first metal layer 240 is made of is chosen from noble metals, preferably gold and silver, metal multilayers, metal oxides and metal multilayers, metal alloys, and has a thickness between 10-70 nm;5. The optical sensor according to anyone of claims 1 to 4 further comprising a first intermediate layer 300 interposed between the sensitive layer 200 and the first metal layer 240, wherein said intermediate layer 300 is made of a material chosen from chromium, titanium, titanium oxide (TiC ), zirconium oxide (ZrC ) and combinations thereof, and has a thickness of between 10-30nm.

6. The optical sensor according to anyone of claims 1 -5 wherein the nanostructured systems of sensitive layer 200 are pollen grains, preferably sunflower grains, preferably having an inter-pollen period of 100 nm to 500 pm and an intra-pollen period of 100 nm to 50 pm.

7. The optical sensor according to anyone of the above claims having a sensitivity in the order of attomolar concentration.

8. The optical sensor according to anyone of the foregoing claims comprising:• the transparent and guiding layer 1000 in glass with a thickness of 1000 pm;• the first base layer 100 in aminosilane;• a nanostructured sensitive layer 200 consisting of sunflower pollen grains having an inter-pollen period of 100 nm to 500 pm and an intra-pollen period of 100 nm to 50 pm;• the first metal layer 240 in gold with a thickness of 45 nm;• the layer of receptor 400 consisting of an estradiolspecific bioreceptor (ER).

9. A method of detecting an analyte using a sensor according to anyone of the above claims, comprising:• irradiating the sensor with electromagnetic radiation;• acquiring the resulting optical spectrum;• determining the wavelength shift of the maximumplasmonic resonance as a consequence of the binding of the analyte to the receptor.

10. An array consisting of a plurality of optical sensors according to anyone of claims 1 -8, wherein these sensors are placed side by side on the same geometric plane, and wherein each optical sensor of the plurality of optical sensors has a layer of receptor 400 specific for the detection of an analyte.

11. The array having the same multilayer structure as the optical sensor according to anyone of claims 1 -8 except for the layer of receptor 400, wherein the surface of the metal layer 240 is not uniformly coated with a single layer of receptor 400 but is coated with a plurality of layers of receptor 400, different from each other, to form distinct surface regions, according to any geometry, each specific to a given analyte.

12. A sensor system for the detection of an analyte in a sample comprising:• at least one optical sensor according to anyone of claims 1 -8 or an array according to anyone of claims 10-1 1 ;• at least one source of electromagnetic radiation, preferably visible;• at least one detection device chosen from spectrophotometers, spectrometers, photodiodes;• at least one reference chip, said at least one reference chip for spectral normalization being identical to at least one optical sensor except for the absence of the sensitive layer 200 and the receptor layer 400;• at least one sample holder.

13. The sensor system according to the above claim further comprising a data processor such as a computer.

14. The sensor system according to anyone of claims 12-13 wherein the at least one source of electromagnetic radiation and the at least one detection device are respectively implemented within at least one first and one second separate optoelectronic board.

15. The sensor system according to anyone of claims 13-14 further comprising computerized means for analysing the data provided by the sensor, in turn comprising at least one processor, one storage memory and means for displaying data.

16. The sensor system according to the above claim wherein the means for displaying data are local, such as a display, or remote such as a computer, smartphone, or other electronic device.

17. A computer program that, when executed, instructs the sensor system according to anyone of claims 13-16 to perform a method for determining an analyte in a sample.

18. A machine-readable storage medium comprising the program according to claim 17.

19. A computer system comprising the computer program in accordance with claim 17, wherein the computer system may optionally be a tablet or a smartphone.

0. A method for determining an analyte in a sample by means of the sensor system according to anyone of claims 12-16 comprising the steps of:• allocating at least one optical sensor and at least one reference chip in the sensor system with a sample holder connected to at least one source and at least one detector;• acquiring the output signals from the optical sensor and the reference chip, obtained by placing said optical sensor and reference chip in contact with or immersed in the medium, said medium being made up of the sample in the absence of the analyte;• normalizing the output signal from the optical sensor with respect to the output signal obtained from the reference chip;• reading the normalized signal;• immersing the optical sensor and reference chip in the sample to be analyzed;• incubating the sample so that the analyte binds to the receptor 400 layer of the optical sensor;• optionally, washing the optical sensor to remove any interferents;• acquiring the output signals from the reference chip and the optical sensor to which the analyte to be examined is bound, said signals being obtained by placing said optical sensor and reference chip in contact with or immersed in the medium deprived of the analyte;• normalizing the output signal from the optical sensor with respect to the output signal obtained from the reference chip;• detecting the variation of the signal with respect to the medium.

21. A portable kit for the determination of an analyte in a sample comprising:• a sensor system according to anyone of claims 12-16;• instructions for use, optionally accessible remotely through an electronic medium.• optionally, computerized means for analysing the data provided by the sensor comprising at least one processor, one storage memory, and means for displaying data.

22. Use of the optical sensor according to anyone of claims 1 -8, or the array according to anyone of claims 9-10 or the sensor system according to anyone of claims 12-16 for the determination of the presence or quantity of an analyte in a sample, wherein such sample may be a fluid chosen from a liquid or gaseous solution, or an aerosol.

23. The use according to the previous claim in an application area chosen from: diagnosis of tumor markers, analysis of various emerging environmental pollutants (in rivers, lakes, seas, agricultural land, wooded areas), bioterrorism or biodefense, detection of viruses and bacteria, pandemic emergencies, personalized medicine, Point-of-care tests for different analyses or pre-analyses, tests on rare diseases, measurement of explosives.

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