Diffractive sensor for sensing target analytes in sample, and system and method for sensing target analytes in sample by that diffractive sensor

Abstract

To make available devices that enable detection of viruses or bacteria, as well as other target analytes, in a sample in a simple, fast, reliable and reproducible manner.SOLUTION: The present invention relates to a diffractive sensor (1) for sensing a target analyte, comprising: a diffractive layer comprising a plurality of surface regions equal to each other and having a maximum dimension included in between 5 μm and 50 μm, each surface region comprising a diffractive grating provided with grooves having a depth less than 200 nm, where in each of the surface regions equal to each other, the diffractive grating has the same conformation; and a receptor layer overlapping the diffractive layer and configured to be selectively coupled to the target analyte. The present invention also relates to a system and method for detection of a target analyte in a sample by means of the diffractive sensor.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a sensor that utilizes the optical phenomenon of diffraction by a diffraction grating to detect a target analyte in a sample. The present invention also relates to a method and system for sensing a target analyte in a sample that utilizes such a diffractive sensor.

[0002] The term "target analyte" refers to any chemical species whose presence in a sample is to be determined.

[0003] The present invention has particular application for detecting target analytes such as viruses or bacteria, or components thereof, but may also be applied to the detection of other types of target analytes and therefore may be applied in the medical, veterinary and diagnostic fields, but also, for example, in the biosafety or chemical fields, particularly for the detection of trace amounts of contaminants. [Background technology]

[0004] Detection of the target analyte can be performed according to a number of criteria and techniques, which depend on the properties of the analyte itself.

[0005] Definitive diagnosis of infection, for example by detection of viral or bacterial infectious agents, is generally performed by more or less complex tests, which usually require the use of sophisticated laboratory equipment and take a long time, up to several days, before the results are available. Alternatively, rapid tests are available, but these have a much lower sensitivity than the tests mentioned above and are therefore not completely reliable and may give questionable results from a clinical point of view.

[0006] There is therefore a particular need to make available devices that allow the detection of viruses or bacteria, as well as other target analytes, in a sample in a simple, rapid, reliable and reproducible manner.

[0007] For example, EP 3907507 A1 describes a colorimetric sensor comprising a functional layer comprising a nanomaterial capable of generating surface plasmons that are bioresponsive to bacteria and / or viruses, a receptor layer comprising a protein substance or antibody that acts as a virus receptor, and a plasmonic nanostructured layer comprising etched nanostructures such that a plasmonic color is generated. When secretions from an individual containing viruses or bacteria are placed in contact with the functional sensor layer, a change in the structure of the functional sensor layer causes a change in the plasmonic energy and thus a change in the perceived color. A change in coloration thus indicates the presence of viruses or bacteria in the secretions. Summary of the Invention

[0008] It is an object of the present invention to provide an alternative sensor to sensors of known technology, which allows simple, rapid and reliable detection of common target analytes such as bacteria or viruses.

[0009] This and other objects are achieved by a diffraction sensor for sensing a target analyte according to claim 1, a system for sensing a target analyte according to claim 17 and a method for sensing a target analyte according to claim 22.

[0010] The dependent claims define possible advantageous embodiments of the invention. [Brief description of the drawings]

[0011] In order to better understand the invention and to realize its advantages, some non-limiting exemplary embodiments thereof will now be described with reference to the accompanying drawings, in which: [Figure 1] 1 is a schematic diagram of a diffraction sensor according to an embodiment of the present invention; [Diagram 2] 1 is a close-up view of a diffractive layer of a diffractive sensor according to an embodiment in which possible adjacent surface areas are highlighted. [Diagram 3] 4 shows an enlarged view of a diffractive layer of a diffractive sensor according to a further embodiment, highlighting possible surface areas that overlap one another; [Figure 4] FIG. 2 is a close-up view of a possible surface area of ​​a diffractive layer of a diffractive sensor according to an embodiment. [Diagram 5] FIG. 1 is a schematic diagram of a system for sensing a target analyte in a sample, according to one embodiment. [Figure 6] 3 shows possible diffraction images produced by a diffraction sensor according to the invention; [Figure 7] Figures 7a) and 7b) show two possible diffraction images generated by a diffraction sensor in the absence and presence, respectively, of a target analyte in a sample applied to the diffraction sensor itself. [Figure 8] 8a) to 8d) are schematic diagrams of an antibody in which the -SS disulfide bond has been reduced to a reduced -SH disulfide bond and a fraction thereof, respectively. [Figure 9] FIG. 8B is a schematic diagram of the antibody fraction and target antigen shown in FIG. 8b) bound to a receiving layer of a diffractive sensor according to one embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] 1 to 4 of the accompanying drawings, a diffractive sensor for sensing a common target analyte (or, as will be understood, multiple target analytes) is generally referred to as 1. The sensor 1 may, for example, be made in the form of a label that is affixed to a user device (not shown).

[0013] The sensor 1 preferably includes a support layer 2, the function of which is to support additional upper layers, as will be described in detail below. The support layer 2 is preferably transparent or translucent and may be made of, but is not limited to, polycarbonate, or PVC, or Teslin, or polyester, or the like.

[0014] The sensor 1 preferably comprises a diffractive layer 3 applied directly or indirectly to a support layer 2 .

[0015] The diffractive layer 3 comprises a diffraction grating 30 which is nanostructured, ie provided with diffractive structures having a depth of the order of tens to hundreds of nanometers, as will be described in more detail below.

[0016] The diffraction grating 30 can be realized as follows. on a polymer film, such as a polyester film (polycarbonate PC, polyethylene terephthalate PET, polyvinyl chloride PVC, polypropylene PP, etc.), preferably having a thickness comprised between 5 μm and 500 μm; of amorphous (for example glass) or highly crystalline (for example quartz) oxide materials, preferably with a thickness comprised between 10 μm and 500 μm; -Made of fiberglass material.

[0017] For example, the diffraction grating 30 may be fabricated by one of the following techniques. ablation: this technique involves the selective removal of material and includes, for example, pulsed laser techniques such as the LIPSS (Laser-induced periodic surface structures) technique, or the LEHCEB (Low Energy High Current Electron Beam) technique; - Deposition: this technique involves the deposition of a thin material onto a surface and includes, for example, sputtering and optical photolithography.

[0018] The diffractive layer 3 comprises a number of surface areas 40 which are identical to one another, i.e. the diffraction gratings 30 have the same structure. Furthermore, the surface areas 40 have the same shape and dimensions.

[0019] The surface areas 40 may have any shape and, according to a possible embodiment, they have a square contour. The surface areas 40 have a maximum dimension (identifiable as the maximum distance between two points of the contour and which corresponds to one side of the square in the case of a cell with a square contour) preferably comprised between 5 μm and 50 μm, even more preferably between 30 μm and 45 μm, for example equal to 40 μm.

[0020] The surface regions 40 may be arranged side by side (e.g., in the case of a square surface region, they may have a checkerboard arrangement as shown in FIG. 2) and / or may partially overlap each other (in other words, the vertices of one surface region may be included in another surface region, as shown in FIG. 3).

[0021] Referring now to a single surface region 40 (FIG. 4), the diffraction grating 30 has grooves that form a pattern that is preferentially random, but which repeats equally in each surface region 40. As can be seen in FIG. 4, where the darker areas represent the valleys and the lighter areas represent the peaks of the grooves of the diffraction grating 30, no geometrically defined pattern formed by the grooves can be discerned (in this sense, the pattern of grooves of the diffraction grating 30 has a "random" pattern).

[0022] The groove depth of the diffraction grating 30 (i.e. the distance between the peaks and the valleys) is less than 200 nm, preferably comprised between 30 nm and 200 nm, more preferably between 100 nm and 180 nm. In an embodiment, the diffraction sensor 1 further comprises a protective layer 4 for protecting the diffraction grating 30, the protective layer 4 preferably having a thickness comprised between 1 μm and 100 μm. The protective layer 4 may be made of a material selected from the group consisting of: -Sulfides (e.g. zinc sulfide (ZnS)) -Oxides (such as titanium oxide (TiO2), zinc oxide (ZnO), zirconium oxide (ZrO2) and silicon oxide (SiO2)); -Metals (such as gold (Au), silver (Ag), nickel (Ni), zinc (Zn), aluminum (Al), and copper (Cu)).

[0023] The protective layer 4 is preferably made by depositing (for example under vacuum) nanoparticles of the aforementioned materials on the diffraction grating 30. Preferably, the size of the nanoparticles is comprised between 4 and 30 nm, even more preferably less than or equal to 20 nm, for example equal to 8 nm.

[0024] The diffraction grating 30 of the diffraction layer 3 adapted as described above is such that when a beam of monochromatically polarized light (laser), emitted by a laser source 100 placed behind the sensor 1 itself (i.e. on the side of the sensor where the support layer 2 is located) and preferably incident at 90° passes through the sensor 1, this light beam is diffracted into a diffraction image visible to the naked eye on the screen 200, according to the well-known Bragg law: nλ=2dsinθ During the ceremony, -n is a positive or negative integer (diffraction order), -λ is the wavelength of the laser beam, -d is the distance between the sensor 1 and the screen 200 onto which the diffraction image is projected, −θ is the first order diffraction angle, which depends on the structure of the diffraction grating.

[0025] In other words, due to the structure of the nanometric diffraction grating, the diffraction phenomenon occurs at a macroscopic level, i.e. at a level that can be seen by the naked eye, similar to the phenomena that occur at the atomic level after exposing a material to, for example, X-rays, but which cannot be detected by the naked eye.

[0026] As an example, the laser light emitted by the laser source 100 may have a wavelength λ equal to 532 nm (green light), but the diffraction image may be generated by illuminating the sensor 1 with laser light having any other wavelength in the visible spectrum (directly included between 390 nm and 700 nm).

[0027] An example of a diffraction image produced on the screen 200 is shown in Figure 6. It contains a number of dots, the distribution of which depends on the structure of the diffraction grating 30, which is repeated within the surface area 40. In this figure, a larger dot can be seen, which is aligned with the laser light incident at 90° to the rear of the sensor, and which passes through the sensor undiffracted.

[0028] Given the periodicity of the surface regions 40, which are repeated equally within the diffractive layer 30, it is observed that a diffraction image is produced wherever on the sensor 1 the laser light beam is directed.

[0029] The structure of the protective layer 4 can affect the brightness of the dots in the diffraction image on the screen 200. For example, it has been observed that the use of zinc nanoparticles in the protective layer 4 results in a brighter diffraction image than when gold nanoparticles are used, but the distribution of the dots in the diffraction image is still the same. Thus, the structure of the diffraction grating in the surface region 40 makes the sensor 1 generate a kind of unique fingerprint of the sensor 1, i.e., the diffraction image exactly. Varying the random pattern structure of the diffraction grating 30 also results in a different distribution of the dots in the diffraction image.

[0030] It is observed that the diffractive layer 3 is adapted so that the light diffracted by the sensor is polarized.

[0031] The diffraction image can be obtained not only by traversing the sensor from the rear side with a laser beam as described above, but also by reflecting the laser beam, i.e., by directing the laser beam obliquely toward the front side of the sensor (the side opposite to the bottom side, for example, the side where the protective layer 4 may be disposed). In either case, a diffraction image having the above characteristics can be obtained.

[0032] Returning now to FIG. 1, the diffractive sensor 1 further comprises a receiving layer 5 overlying and in direct contact with the diffractive layer 3 or overlying and in direct contact with the protective layer 4 (if present).

[0033] The receptive layer 5 is capable of selectively binding to the target analyte to be detected and is not capable of binding to substances of a different nature. The target analyte may be contained in a sample, such as a biological solution (e.g. a saliva or blood or urine sample), and may be deposited, e.g. smeared or rubbed, onto the receptive layer 5.

[0034] In this way, if the target analyte is not present in the sample, the receptive layer 5 does not change and the diffraction sensor 1, when exposed to the laser light beam as described above, generates a first diffraction image on the screen that is visible to the naked eye. On the other hand, if the target analyte is present in the sample, the target analyte binds to the receptive layer 5, so that the diffraction sensor 1 generates a second diffraction image that is different from the first diffraction image. This second diffraction image is also visible to the naked eye and is similar to the diffraction image of the first state. Figure 7 shows a comparison of the diffraction images generated in the absence of the target analyte (Figure 7a)) and in the presence and binding of the target analyte to the receptive layer 5 (Figure 7b). As can be seen, the number and / or distribution and / or light intensity of dots visible in the diffraction images in the first and second cases are different. It is therefore possible to determine whether the target analyte is present in the sample and bound to the receptive layer 5 of the diffraction sensor 1 from a simple visual comparison of the two diffraction images, which can also be performed with the naked eye.

[0035] In general, the diffraction image produced by the diffractive layer 3 alone will differ from the diffraction image produced as a result of applying the receptive layer 5, and also from the diffraction image produced when the receptive layer 5 is bound to a target analyte. Thus, a sensor comprising a diffractive layer 3 with or without a protective layer 4, and lacking a receptive layer 5, can therefore produce a diffraction image characteristic of a nanometric diffraction grating that constitutes a kind of fingerprint of the sensor.

[0036] It should be observed that the diffraction images generated in the presence and absence of the target analyte may also differ from each other in coloration. However, this may be a secondary effect on the variation of the diffraction images. Preferably, the diffraction images, whose color depends on the laser light used (for example, for a wavelength λ=532 nm, the color is green), are converted to a gray scale.

[0037] In order that any contaminants other than the target analyte present in the sample and / or in the environment in which the sensor is located do not alter the diffraction image, the receptive layer 5 is preferably washed in a suitable manner following application of the sample in which the presence of a possible target analyte is detected. Some possible examples are given below. In this way, substances or molecules or contaminants that cannot bind to the receptive layer 5 (which can only selectively bind to the target analyte) are removed.

[0038] The nature of the receptive layer 5, specifically its chemical composition, as well as the method of washing (if applicable), may vary depending on the target analyte of interest. In general, the receptive layer 5 may comprise antibodies, proteins, molecules with locking properties, chelating compounds, or other chemical or biological substances with chelating functionality.

[0039] By way of non-limiting example, target analytes may include: - Human chorionic gonadotropin (hCG): in this case the receptive layer 5 may contain a monoclonal antibody specific for hCG and the sensor 1 is suitable for pregnancy testing. Tetanus toxoid: in this case the receptor layer 5 may contain a monoclonal antibody specific for tetanus toxoid, allowing the sensor 1 to detect tetanus toxoid. - Nucleocapsid (N) protein of SARS-CoV-2: in this case, the receptive layer 5 may contain a monoclonal antibody specific for the nucleocapsid protein of SARS-CoV-2, allowing the sensor 1 to detect the presence of the SARS-CoV-2 virus in a biological sample.

[0040] Thus, the inclusion of the above-mentioned antibodies in the receptive layer 5 enables the latter to selectively detect the presence of these target analytes in a sample applied to the receptive layer 5. In general, suitable antibodies may also be specifically raised against the components to be detected by the sensor.

[0041] Typically, if the target analyte is a specific antigen, the receptive layer is specific for said antigen and may contain antibodies non-specific for antigens other than the target antigen intended to be detected by the sensor 1. For reliable detection it is therefore necessary that the antibodies are tightly bound to the diffractive layer 3. Advantageously, such a tight binding can be achieved by binding the antibodies to nanoparticles, preferably metal nanoparticles, of the protective layer 4.

[0042] Antibodies are modular protein complexes with a common basic structure, but show variability in specific regions that allow them to bind specific antigens. In general, antibodies exhibit a Y-shaped conformation with a central stem and two side branches. They contain four chains covalently linked by -SS disulfide bridges and cannot bind metals such as gold unless previously treated.

[0043] One possible pretreatment of the antibody for binding to the protective layer 4, and in particular to the metal nanoparticles thereof, involves cleaving the -SS disulfide bonds of a part of the antibody by reducing them to reduced -SH disulfide bonds, which can instead bind to the metal, in particular form very strong thiol bonds with the metal. If the -SS disulfide bonds of the antibody are cleaved, four different parts of the antibody are obtained (Figures 8a-d), and only half of the whole antibody (shown in Figure 8b), i.e., a single side chain, i.e., the Fab' fragment, and half of the stem, i.e., half of the Fc fragment, are suitable on the one hand to form a stable bond with the metal of the protective layer 4 and on the other hand to maintain the binding ability to the target antigen 10 (Figure 9). The disulfide-SS bonds are then reduced in the hinge region that holds the two heavy chains of the antibody together, separated.

[0044] Since all antibody moieties obtained by cleaving disulfide-SS bonds differ in size, it is possible to select the desired antibody moiety, for example, by precisely filtering according to size.

[0045] Reduction of -SS disulfide bonds Reduction to -SH disulfide bonds can be achieved by molecular techniques known per se (in particular, the use of reducing agents, polyacrylamide gels, ultrafiltration devices, etc.). In this regard, see, for example, the following papers, which are fully incorporated herein by reference: -Rafael Antonio Salinas Dominguez, Miguel Angel Dominguez Jimenez, Abdu Orduna Diaz: "Antibody Immobilization in Zinc Oxide Thin Films as an Easy-Handle Strategy for Escherichia coli Detection" - ACS Omega 2020,5,20473-20480; -Anke K. Trilling, Jules Beekwildera, Han Zuilhof: "Antibody orientation on biosensor surfaces: a minireview" - Analyst, 2013, 138, 1619; -Lu Zhang, Yacine Mazouzi, Michele Salmain, Bo Liedberg, Souhir Boujday: "Antibody-Gold Nanoparticle Bioconjugates for Biosensors: Synthesis, Characterization and Selected Applications" - Biosensors and Bioelectronics 165 (2020)112370; -Asta Makaraviciute,Carolyn D.Jackson,Paul A.Millner,Almira Ramanaviciene: "Considerations in producing preferentially reduced half-antibody fragments" -Journal of Immunological Methods 429(2016)50-56; -Monalisa Pal,Sanghee Lee,Donghoon Kwon,Jeongin Hwang,Hyeonjeong Lee,Seokyung Hwang,Sangmin Jeon:「Direct immobilization of antibodies on Zn-doped Fe3O4 nanoclusters for detection of pathogenic bacteria」-Analytica Chimica Acta 952(2017)81-87; -Md. Morsaline Billah,Christopher S.Hodges,Henry C.W.Hays,P.A. Millner:「Directed immobilization of reduced antibody fragments onto a novel SAM on gold for myoglobin impedance immunosensing.」-Bioelectrochemistry 80(2010)49-54; -Hongcheng Liu & Kimberly May(2012):「Disulfide bond structures of IgG molecules」-mAbs,4:1,17-23; -Roberto Reverberi,Lorenzo Reverberi:「Factors affecting the antigen-antibody reaction」-Blood Transfus 2007;5:227-240; -Harsh Sharma,Raj Mutharasan:「Half Antibody Fragments Improve Biosensor Sensitivity without Loss of Selectivity」-Anal. Chem. 2013,85,2472-2477; -Woochang Lee,Byung-Keun Oh,Won Hong Lee,Jeong-Woo Choi:「Immobilization of antibody fragment for immunosensor application based on surface plasmon resonance.」-Colloids and Surfaces B:Biointerfaces 40(2005)143-148; -Yuanli Song,Hui Cai,Zhijun Tan,Nesredin Mussa,Zheng-Jian Li:「Mechanistic insights into inter-chain disulfide bond reduction of IgG1 and IgG4 antibodies」-Applied Microbiology and Biotechnology(2022)106:1057-1066; -Nicholas G.Welch,Judith A.Scoble,Benjamin W.Muir,et al:「Orientation and characterization of immobilized antibodies for improved immunoassays」-Biointerphases 12,02D301(2017); -Arkady A.Karyakin,Galina V.Presnova,Maya Yu.Rubtsova,Alexey M.Egorov:「Oriented Immobilization of Antibodies onto the Gold Surfaces via Their Native Thiol Groups」-Anal. Chem. 2000,72,3805-3811; -Nur Mustafaoglu, Tanyel Kiziltepe, Basar Bilgicer: "Site-Specific Conjugation of Antibody on Gold Nanoparticle Surface for One-Step Diagnosis of Prostate Specific Antigen with Dynamic Light Scattering" -Nanoscale.2017 June 29;9(25):8684-8694; -Harry W Schroeder Jr, Lisa Cavacini: "Structure and Function of Immunoglobulins" -Allergy Clin Immunol.2010 February;125(2 0 2):S41-S52.

[0046] If the receptive layer 5 comprises antibodies or other proteins, such washing can be performed, for example, by using PBS (phosphate buffered saline), by immersion and optionally subsequent centrifugation of the sensor.

[0047] It should be noted that according to one possible embodiment, the receptive layer 5 is configured to selectively bind to a plurality of different target analytes, e.g., at different regions of the receptive layer 5, such that different diffraction patterns are generated depending on the bound target analytes. For example, the receptive layer 5 may include different antibodies positioned in such a way that the receptive layer 5 is capable of selectively binding to different target antigens.

[0048] Although the diffraction images generated by the sensor are visible to the naked eye, these diffraction images can be usefully detected by automatic or partially automatic systems. As an example, a possible system for sensing a target analyte in a sample is shown in FIG. 5. The system comprises a laser source 100, a screen 200 (preferably satin) and a vision system capable of acquiring the diffraction images generated by the sensor 1, for example a video camera 300 directed at the screen 200 connected to a control unit of a computer 400. The latter control unit can possibly perform a grayscale conversion of the diffraction images. Such a system can be portable, for example to perform on-site analysis. In such a case, the above-mentioned equipment can be provided with a power source and / or a battery.

[0049] According to a further aspect of the present invention, a method for sensing a target analyte in a sample comprises the following steps. - providing a diffraction sensor 1 according to the above embodiment; - applying a sample to the receiving layer 5 of the diffraction sensor 1; - directing a beam of laser light, e.g. emitted by a light source 100, onto the diffraction sensor 1, such that the diffraction sensor 1 produces a diffraction image visible to the naked eye, e.g. on a screen 200; - comparing the diffraction image generated by the diffraction sensor 1 with a reference diffraction image obtained by shining an equal beam of laser light on the diffraction sensor in the absence of target analytes on the receptive layer 5 (this comparison can be performed by a naked eye operator or, preferably, by an automatic control unit, for example a computer 400); - determining the presence of the target analyte in the sample if the diffraction pattern produced by the diffraction sensor 1 differs from the reference diffraction pattern.

[0050] In one embodiment, the method converts the diffraction image to greyscale to remove coloration that is dependent on the wavelength of the laser light beam. This step may be performed, for example, by the control unit.

[0051] If the receptive layer is capable of selectively binding to multiple target analytes, the method may further include the additional step of comparing the diffraction image generated by the sensor with multiple stored diffraction images (each corresponding to a particular target analyte) and determining the presence of a particular target analyte from the multiple target analytes detectable by the sensor if the diffraction image generated by the sensor matches the stored diffraction image for that particular target analyte.

[0052] In one embodiment, after the step of applying the sample to the receiving layer 5 and before the step of directing the laser light beam to the diffraction sensor 1, the method comprises an additional step of washing the diffraction sensor 1 to remove substances or molecules other than the target analyte or contaminants from the receiving layer 5 that may interfere with the final diffraction image, even if only as background noise. Such a washing step may be performed, for example, by using a buffer solution (e.g., PBS), an ionic or non-ionic detergent, or by a mild surfactant, following appropriate procedures and timing.

[0053] The diffraction sensors, systems and methods according to the present invention may find application in a variety of fields, some non-limiting examples of which are given below. -Diagnosis in a medical or veterinary setting: Diagnosis of bacterial (e.g., Lyme disease, brucellosis, syphilis), viral (e.g., HIV, hepatitis A, B, C), and fungal (e.g., pathogenic yeast infections) infections by demonstration of the pathogen or its components; ○ Virus detection (Coronavirus, HIV, Hepatitis, Ebola, Norovirus, Influenza, West Nile, Zika, etc.); o Diagnosis of autoimmune diseases caused by the abnormal production by the immune system of autoantibodies that destroy cells in the body, by demonstration of specific clinically validated markers (e.g. autoantibodies that destroy insulin-producing cells in the pancreas in type I diabetes); o Detection and quantification of hormones (e.g., human chorionic gonadotropin (hCG), follicle-stimulating hormone (FSH), testosterone); ○ Screening of blood donations (e.g. for detection of viral infections such as HIV); o Detection of drugs (e.g. amphetamines, cocaine); Detection of tumor markers (e.g., prostate-specific antigen (PSA) for diagnosing prostate cancer); Veterinary diagnostics (e.g. detection of African swine fever, avian fever, bovine parvovirus, canine adenovirus, coronavirus, equine infectious anemia, feline leukemia, etc.). -DNA search; -Non-diagnostic uses: Biosafety: ■Communicable disease surveillance; ■ Biological weapons / bioterrorism testing; ■ Testing in cosmetics (e.g. testing for metal contamination); Contaminant detection: ■ Detection and screening of contaminants in foods (e.g., Salmonella spp., Escherichia coli, Campylobacter spp., Staphylococcus aureus); ■ Detection and screening of environmental contaminants (e.g. detection of heavy metals in water or soil); ■ Pesticide detection; ■ Detection of explosives (e.g. detection of nitrates by certain enzymes such as nitrate reductase).

[0054] It should be noted that, as used herein and in the appended claims, the term "overlapping" referring to layers of sensor 1 is not necessarily intended to imply direct contact between the referenced overlapping layers. Thus, such layers may be in direct contact with one another or may have one or more intermediate layers disposed therebetween without compromising their overlap.

[0055] It should also be noted that for purposes of this specification and the appended claims, unless otherwise indicated, all numbers expressing quantities, measures, percentages, and the like, are to be considered in all instances as modified by the term "about." Moreover, all ranges include any combination of the maximum and minimum points disclosed, and include any intermediate ranges within those ranges, whether or not they are specifically recited.

[0056] The present disclosure may be implemented according to at least one of the above aspects and according to one or more of the described embodiments, optionally in combination with each other.

[0057] For purposes of this specification and the appended claims, the terms "an" or "one" should be read as including one or at least one, and the singular forms include the plural forms unless it is clear that otherwise is intended.

[0058] Given the above description of the diffraction sensor and systems and methods for sensing a target analyte in a sample, those skilled in the art may make numerous additions, modifications, or substitutions of elements with functionally equivalent elements to meet specific contingent needs without departing from the scope of the appended claims.

Claims

1. A diffractive sensor (1) for sensing a target analyte, comprising: a diffractive layer (3) comprising a plurality of surface regions (40) which are equal to one another and have a maximum dimension comprised between 5 μm and 50 μm, each surface region (40) comprising a grooved diffraction grating (30) having a depth of less than 200 nm, in each of said surface regions (40) which are equal to one another, said diffraction grating (30) having an identical structure; a receptive layer (5) overlying said diffractive layer (3) and adapted to selectively bind to said target analytes.

2. Diffraction sensor (1) according to claim 1, wherein the grooves of the diffraction grating (30) in each surface region (40) form a pattern having a random tendency.

3. Diffraction sensor (1) according to claim 1 or 2, wherein each of said surface areas (40) has a maximum dimension comprised between 30 μm and 45 μm.

4. Diffraction sensor (1) according to any one of claims 1 to 3, wherein the surface area (40) has a square shape and the maximum dimension is one side of the square.

5. Diffraction sensor (1) according to any one of the preceding claims, wherein the surface areas (40) are arranged side by side.

6. Diffraction sensor (1) according to any one of the preceding claims, wherein the surface areas (40) are partially overlapping.

7. Diffraction sensor (1) according to any one of claims 1 to 6, wherein the grooves of the diffraction grating (30) of each surface region (40) of the diffraction layer (3) have a depth comprised between 30 nm and 200 nm, preferably between 100 nm and 180 nm.

8. The diffractive layer (3) is a polymer film selected from the group consisting of polycarbonate, polyethylene terephthalate, polyvinyl chloride, polypropylene, having a thickness comprised between 5 μm and 500 μm, or - an amorphous or crystalline material having a thickness comprised between 10 μm and 500 μm, or A diffraction sensor (1) according to any one of claims 1 to 7, comprising a glass fibre material.

9. Diffraction sensor (1) according to any one of the preceding claims, further comprising a protective layer (4) for protecting the diffraction grating (30) overlapping and in direct contact with the diffraction layer (3).

10. The protective layer (4) is - Sulfur, or an oxide selected from the group consisting of titanium oxide, zinc oxide, zirconium oxide, silicon oxide, or A diffraction sensor (1) according to claim 9, made of a metal selected from the group consisting of gold, silver, nickel, zinc, aluminium, copper.

11. Diffraction sensor (1) according to claim 9 or 10, wherein the protective layer (4) comprises nanoparticles deposited on the diffraction grating (30) of the diffraction layer (3).

12. Diffraction sensor (1) according to claim 11, wherein the nanoparticles of the protective layer (4) have a size comprised between 4 and 30 nm, preferably less than 20 nm.

13. Diffraction sensor (1) according to any one of claims 9 to 12, wherein the receiving layer (5) overlaps and is in direct contact with the protective layer (4).

14. Diffraction sensor (1) according to any one of the preceding claims, wherein the target analyte is a specific antigen and the receptor layer (5) comprises an antibody adapted to bind to the specific antigen.

15. Diffraction sensor (1) according to claim 14, wherein the antibody comprises a Fab' antibody fraction and half of the Fc antibody fraction, and the disulfide bond -S-S- is reduced to a reduced disulfide bond -SH adapted to be bound to the protective layer (4).

16. Diffraction sensor (1) according to any one of the preceding claims, further comprising a transparent or semi-transparent support layer (2), said diffractive layer (3) overlying said support layer (2).

17. 1. A system for detecting a target analyte, comprising: - a diffraction sensor (1) according to any one of claims 1 to 16, a source (100) of a laser light beam having a wavelength comprised in the visible spectrum, arranged so that said laser light beam is directed onto said diffraction sensor (1) to generate a diffraction image; a screen (200) arranged at a distance from the diffraction sensor (1) and arranged so that the diffraction image generated by the diffraction sensor is projected.

18. 18. The system of claim 17, wherein the laser light source (100) is positioned behind the diffraction sensor (1), such that the laser light beam hits the sensor at 90° and the diffraction image is generated by the laser light beam traversing the diffraction sensor (1).

19. 18. The system of claim 17, wherein the laser light source (100) is positioned in front of the sensor (1), whereby the laser light beam impinges on the diffraction sensor (1) obliquely and the diffraction image is generated by reflection from the diffraction sensor (1).

20. The system according to any one of claims 17 to 19, further comprising a vision system (300) capable of detecting the diffraction image projected on the screen (200).

21. The system of claim 20, further comprising a control unit (400) operably connected to the vision system (300) and configured to compare the diffraction image projected onto the screen (200) with a reference diffraction image and determine the presence of the target analyte on the receiving layer (5) of the diffraction sensor (1) if the detected diffraction image differs from the reference diffraction image.

22. 1. A method for detecting a target analyte in a sample, comprising: - providing a diffraction sensor (1) according to any one of claims 1 to 17, - applying the sample to the receiving layer (5) of the diffraction sensor (1); - directing a laser light beam having a wavelength included in the visible spectrum onto said diffraction sensor (1), whereby said diffraction sensor (1) produces a diffraction image visible to the naked eye; - comparing the diffraction image generated by the diffraction sensor (1) with a reference diffraction image obtained by directing an equivalent laser light beam onto the diffraction sensor (1) but without the target analyte on the receiving layer (5); - determining the presence of the target analyte in the sample if the diffraction image produced by the diffraction sensor (1) differs from the reference diffraction image.

23. 23. The method according to claim 22, comprising, after the step of applying the sample to the receiving layer (5) and before the step of directing a laser light beam to the diffraction sensor (1), a step of cleaning the diffraction sensor (1) to remove contaminants or molecules or agents different from the target analyte from the receiving layer (5).

24. The receptive layer (5) of the diffractive sensor (1) is capable of selectively binding to a plurality of target analytes, and the method comprises the steps of: - comparing the diffraction image generated by the diffraction sensor (1) with a plurality of stored diffraction images, each corresponding to a particular target analyte among the plurality of target analytes; - determining the presence of a particular target analyte of the plurality of target analytes if the diffraction image produced by the diffraction sensor matches the stored diffraction image of the particular target analyte.

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