Diffraction sensors, systems, and methods for target analytes

Abstract

The invention relates to diffraction sensors, systems, and methods for target analytes. Diffraction sensor (1) for sensing a target analyte, comprising:-a diffractive layer (3) comprising a plurality of surface regions (40) equal to one another and having a maximum dimension comprised between 5 [mu] m and 50 [mu] m, each surface region (40) comprising a diffraction grating (30) provided with a groove having a depth of less than 200 nm, where in each of the surface regions (40) equal to one another, the diffraction grating (30) is provided with a groove having a depth of less than 200 nm; the diffraction gratings (30) have the same conformation; -an acceptor layer (5) overlapping the diffractive layer (3), configured to selectively bind to the target analyte. The invention also relates to a system and a method for detecting a target analyte in a sample by means of said diffraction sensor (1).

Description

Technical Field

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

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

[0003] The invention has particular application in the detection of target analytes, such as viruses or bacteria or components thereof, but can also be applied to the detection of other kinds of target analytes, thus not only in the medical, veterinary and diagnostic fields, but also, for example, in the biosafety or chemical fields, in particular for the detection of trace contaminants. Background Art

[0004] Detection of target analytes can be performed according to a variety of criteria and techniques, depending on the nature of the analyte itself.

[0005] For example, reliable diagnosis of infections by detection of viral or bacterial infectious agents is usually performed by more or less complex tests, which usually require the use of complex laboratory equipment and require a long time, even on the order of several days, before the results are obtained. Alternatively, rapid tests are also available, but these have a much lower sensitivity than the above tests and can therefore lead to results that are not completely reliable and are problematic from a clinical point of view.

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

[0007] For example, EP 3907507A1 describes a colorimetric sensor comprising a functional layer of nanomaterials capable of generating surface plasmas that are biologically responsive to bacteria and / or viruses, a receptor layer comprising proteinaceous substances or antibodies that act as viral receptors, and a plasma nanostructure layer comprising etched nanostructures such that plasma colors are generated. When secretions from an individual containing viruses or bacteria are placed in contact with the functional sensor layer, changes in the structure of the functional sensor layer result in changes in plasma energy and therefore in changes in perceived color. Thus, changes in coloration indicate the presence of viruses or bacteria in the secretions. Summary of the invention

[0008] The object of the present invention is to provide an alternative sensor to those according to the known art, which sensor is capable of simple, rapid and reliable detection of common target analytes, such as bacteria or viruses.

[0009] This object 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 appreciate its advantages, some non-limiting exemplary embodiments thereof will be described below with reference to the accompanying drawings, in which:

[0012] Figure 1 is a schematic diagram of a diffraction sensor according to an embodiment of the present invention;

[0013] Figure 2 is an enlarged view of a diffraction layer of a diffraction sensor according to an embodiment, wherein possible side-by-side surface areas are highlighted;

[0014] Figure 3 is an enlarged view of a diffraction layer of a diffraction sensor according to a further embodiment, wherein possible surface areas that partially overlap each other are highlighted;

[0015] Figure 4 is an enlarged view of a possible surface area of ​​a diffraction layer of a diffraction sensor according to an embodiment;

[0016] Figure 5 is a schematic diagram of a system for sensing a target analyte in a sample according to an embodiment;

[0017] Figure 6 shows a possible diffraction image produced by a diffraction sensor according to the present invention;

[0018] Figure 7 A and Figure 7 b shows two possible diffraction images generated by the diffraction sensor in the absence and presence of the target analyte in the sample applied to the diffraction sensor itself, respectively;

[0019] Figure 8 a to Figure 8 d are schematic diagrams of an antibody and its fractions in which a -SS-disulfide bond is reduced to a reduced -SH disulfide bond;

[0020] Fig. 9 yes Figure 8 Schematic diagram of the antibody fraction bound to the receptor layer of the diffraction sensor according to the embodiment and the receptor layer of the target antigen shown in b. DETAILED DESCRIPTION

[0021] Reference Figures 1 to 4 , a diffraction sensor for sensing a generic target analyte (or, as will be seen, a plurality of target analytes) is generally referred to as 1. The sensor 1 may be made, for example, in the form of a tag to be fixed to a user instrument (not shown in the figures).

[0022] The sensor 1 preferably comprises a support layer 2, which has the function of supporting an additional cover layer, as will be described in detail below. The support layer 2 is preferably transparent or translucent, and can be made of, but not limited to, polycarbonate, PVC, Teslin, polyester, and the like.

[0023] The sensor 1 comprises a diffractive layer 3 which is preferably applied directly or indirectly to a support layer 2 .

[0024] The diffraction layer 3 comprises a nanostructured diffraction grating 30, ie a diffraction structure having a depth of approximately several tens to several hundreds of nanometers is provided, as will be described in detail below.

[0025] The diffraction grating 30 may be implemented as:

[0026] on a polymer film, such as a polyester film (such as polycarbonate PC, polyethylene terephthalate PET, polyvinyl chloride PVC, polypropylene PP), preferably with a thickness comprised between 5 μm and 500 μm;

[0027] - in amorphous (eg glass) or highly crystalline (eg quartz) oxide materials, the thickness is preferably comprised between 10 μm and 500 μm;

[0028] -In fiberglass materials.

[0029] For example, the diffraction grating 30 may be made by one of the following techniques:

[0030] - Ablation: this technique involves the selective removal of material and includes, for example, pulsed laser techniques such as the LIPSS (Laser Induced Periodic Surface Structure) technique or the LEHCEB (Low Energy High Current Electron Beam) technique;

[0031] - Deposition: This technique involves depositing thin materials on a surface and includes, for example, sputtering and optical lithography.

[0032] The diffractive layer 3 comprises a plurality of surface areas 40 which are equal to one another, ie in which the diffraction gratings 30 have the same configuration. Furthermore, the surface areas 40 have the same shape and size.

[0033] The surface areas 40 may have any shape and according to a possible embodiment they have a square outline. The surface areas 40 preferably have a maximum dimension (identifiable as the maximum distance between two points of the outline, coinciding with a single side of the square in the case of a square outline unit) comprised between 5 μm and 50 μm, more preferably still between 30 μm and 45 μm (e.g. equal to 40 μm).

[0034] The surface areas 40 may be arranged side by side (for example, in the case of square surface areas, they may have a checkerboard arrangement, such as Figure 2 ), and / or they may partially overlap each other (in other words, a vertex of one surface region may fall within another surface region, as shown in FIG. Figure 3 shown).

[0035] Now referring to the single surface area 40 ( Figure 4 ), the diffraction grating 30 has grooves which form a pattern which preferentially has a random pattern, which however is repeated equally in each surface area 40. Figure 4 As can be seen in the figure, where the darker parts represent the valleys of the grooves of the diffraction grating 30 and the lighter parts represent the peaks of the grooves, no geometrically defined pattern formed by the grooves can be identified (in this sense, the pattern of grooves of the diffraction grating 30 has a "random" pattern).

[0036] The groove depth (i.e., the distance between peaks and valleys) of the diffraction grating 30 is less than 200 nm, preferably comprised between 30 nm and 200 nm, and 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, which preferably has 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:

[0037] - Sulfides, such as zinc sulfide (ZnS)

[0038] - oxides, such as titanium oxide (TiO2), zinc oxide (ZnO), zirconium oxide (ZrO2) and silicon oxide (SiO2);

[0039] -Metals such as gold (Au), silver (Ag), nickel (Ni), zinc (Zn), aluminum (Al) and copper (Cu).

[0040] The protective layer 4 is preferably made by depositing (for example under vacuum) nanoparticles of the above material on the diffraction grating 30. Preferably, the size of the nanoparticles is comprised between 4 nm and 30 nm, more preferably less than or equal to 20 nm, for example equal to 8 nm.

[0041] The diffraction grating 30 of the conformal diffraction layer 3 as described above is such that, if a monochromatic polarized light (laser) beam 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, the beam is diffracted into a diffraction image visible to the naked eye on the screen 200, without the need to insert (between the laser and the sensor, and between the sensor and the screen) optical devices, such as filters or lenses, to magnify the image according to the well-known Bragg law ( Figure 5 ):

[0042] nλ=2dsinθ

[0043] in:

[0044] -n is a positive or negative integer (diffraction order);

[0045] -λ is the wavelength of the laser beam;

[0046] -d is the distance between the sensor 1 and the screen 200 onto which the diffracted image is projected

[0047] -θ is the 1st order diffraction angle, which depends on the configuration of the diffraction grating.

[0048] In other words, due to the conformation of the nano-diffraction grating, the diffraction phenomenon occurs on a macroscopic level, ie visible to the naked eye, similar to what occurs on an atomic level, for example, after a substance is exposed to X-rays, which, however, is not detectable to the naked eye.

[0049] As an example, the laser light emitted by the laser source 100 may have a wavelength λ equal to 532 nm (green light), however, the diffraction image may be generated by irradiating the sensor 1 with laser light having any other wavelength in the visible spectrum, indicatively comprised between 390 nm and 700 nm.

[0050] An example of a diffraction image produced on screen 200 is shown in FIG. Figure 6 It comprises a plurality of spots whose distribution depends on the conformation of the diffraction grating 30 repeated in the surface area 40. In this figure, a larger spot can be seen, which is the spot aligned with the laser light incident at 90° behind the sensor and passes through it without being diffracted.

[0051] It can be observed that, given the periodicity of the surface areas 40 which are equally repeated in the diffractive layer 30 , a diffraction image is generated wherever the laser beam is aimed on the sensor 1 .

[0052] The conformation of the protective layer 4 can affect the brightness of the points 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 if gold nanoparticles were used, but the distribution of the points in the diffraction image remains the same. Thus, the conformation of the diffraction grating in the surface area 40 causes the sensor 1 to produce a unique fingerprint of the sensor 1, i.e., precisely, the diffraction image. Changing the random pattern conformation of the diffraction grating 30 will also result in a different distribution of the points in the diffraction image.

[0053] It can be observed that the diffractive layer 3 is conformal so that the light diffracted by the sensor is polarized.

[0054] It should also be noted that, as described above, a diffraction image can be obtained not only by passing the laser beam through the sensor from the back side, but also by reflection of the laser beam, that is, by directing the laser beam at a certain angle to the front surface of the sensor (that is, on the side opposite to the bottom surface, for example, on the side where the protective layer 4 can be arranged). In both cases, a diffraction image having the above-mentioned characteristics is obtained.

[0055] Now return to Figure 1 The diffraction sensor 1 further comprises a receptor layer 5 , which overlaps with and is in direct contact with the diffraction layer 3 or, if present, overlaps with and is in direct contact with the protective layer 4 .

[0056] The receptor layer 5 can selectively bind to the target analyte to be detected without binding to substances of different properties. The target analyte can be contained in a sample, such as a biological solution (e.g., saliva or blood or urine sample), which can be deposited (e.g., smeared or rubbed) on the receptor layer 5.

[0057] In this way, if the target analyte is not present in the sample, the receptor layer 5 is not changed, and the diffraction sensor 1 generates a first diffraction image visible to the naked eye on the screen when subjected to the laser beam as described above. On the other hand, when the target analyte is present in the sample, it is bound to the receptor layer 5, and therefore, the diffraction sensor 1 generates a second diffraction image different from the first diffraction image, which is also visible to the naked eye and is comparable to the diffraction image of the first condition. Figure 7 In the absence of target analyte ( Figure 7 a) and in the case where the target analyte is present and bound to the receptor layer 5 ( Figure 7 b) Comparison between the diffraction images produced by the first and second cases. It can be seen that the number and / or distribution and / or light intensity of points visible in the diffraction images of the first and second cases are different. Therefore, from a simple visual comparison of the two diffraction images (which can also be performed with the naked eye), it can be determined whether the target analyte is present in the sample and has bound to the receptor layer 5 of the diffraction sensor 1.

[0058] Generally speaking, the diffraction image produced by the diffraction layer 3 alone is different from the diffraction image produced due to the application of the receptor layer 5, and is also different from the diffraction image produced when the receptor layer 5 is bound to the target analyte. Therefore, a sensor including the diffraction layer 3, with or without the protective layer 4, and lacking the receptor layer 5 is also able to produce a diffraction image that is unique to the nano-diffraction grating, which constitutes a kind of fingerprint of the sensor.

[0059] It should be observed that the diffraction images produced in the presence and absence of the target analyte may also differ from each other in terms of coloration. However, this is a possible secondary effect on the variation of the diffraction image. Preferably, the diffraction image whose color depends on the laser used (e.g., for wavelength λ=532 nm, the color is green) is converted into grayscale.

[0060] It should also be noted that in order to prevent any contaminants other than the target analyte present in the sample and / or in the environment in which the sensor is located from changing the diffraction image, it is preferred that, after applying the sample to be detected for the possible presence of the target analyte, the receptor layer 5 is washed in a suitable manner, some possible examples of which are given below. In this way, substances or molecules or contaminants that cannot bind to the receptor layer 5 (which can selectively bind only to the target analyte) are removed.

[0061] The nature of the receptor layer 5, in particular its chemical composition, and the washing method (if applicable) may vary depending on the target analyte sought. Generally speaking, the receptor layer 5 may include antibodies, proteins, molecules with a keying effect, chelating compounds or other chemical or biological substances with a chelating function.

[0062] By way of non-limiting example, target analytes may include:

[0063] - Human chorionic gonadotropin (hCG): in this case, the receptor layer 5 may comprise monoclonal antibodies specific for hCG and the sensor 1 will be suitable for pregnancy testing;

[0064] - Tetanus toxoid: in this case, the receptor layer 5 may comprise monoclonal antibodies specific for tetanus toxin and the sensor 1 will be able to detect tetanus toxin;

[0065] -Nucleocapsid (N) protein of SARS-CoV-2: In this case, the receptor layer 5 may include monoclonal antibodies specific for the nucleocapsid protein of SARS-CoV-2, and the sensor 1 will be able to detect the presence of the SARS-CoV-2 virus in a biological sample.

[0066] Therefore, the inclusion of the above mentioned antibodies in the receptor layer 5 enables the latter to selectively detect the presence of these target analytes in a sample applied to the receptor layer 5. Generally speaking, suitable antibodies may also be generated specifically for the components to be detected by the sensor.

[0067] Generally speaking, in the case where the target analyte is a specific antigen, the receptor layer may include an antibody specific for the antigen and non-specific for antigens other than the target antigen, which is intended to be detected by the sensor 1. Therefore, for reliable detection, the antibody must be firmly bound to the diffractive layer 3. Advantageously, this strong binding can be achieved by binding the antibody to nanoparticles (preferably metal nanoparticles) of the protective layer 4.

[0068] Antibodies are modular structured protein complexes that have a common basic structure but show variability in specific regions that are capable of binding to specific antigens. Generally speaking, antibodies exhibit a Y conformation with a central stem and two side branches. They contain four chains covalently linked by -SS- disulfide bridges and cannot bind to metals such as gold unless pre-processed.

[0069] One possible pretreatment of the antibody for the purpose of binding it to the protective layer 4, in particular to the metal nanoparticles of the protective layer, involves breaking the -SS-disulfide bonds of a part of the antibody by reducing them to reduced -SH disulfide bonds, which in turn are able to bind to the metal, in particular to form very strong thiol bonds with the metal. If the -SS-disulfide bonds of the antibody are broken, four different parts of the antibody are obtained ( Figure 8 a to Figure 8 d), where only half of the intact antibody (in Figure 8 b), i.e., a portion of the antibody comprising a single side branch, i.e., a Fab' fragment, and half of the stem, i.e., half of the Fc fragment, which is suitable for forming a stable bond with the metal of the protective layer 4 on the one hand, and for maintaining the ability to bind to the target antigen 10 on the other hand ( Fig. 9 The -SS-disulfide bonds are then reduced in the hinge region that holds the two separate heavy chains of the antibody together.

[0070] Since all antibody parts obtainable by cleaving the -SS-disulfide bonds are of different sizes, the desired antibody part can be selected, for example, by precise filtering according to size.

[0071] The reduction of -SS-disulfide bonds to reduced -SH disulfide bonds can be accomplished by molecular techniques known per se (such as, in particular: using reducing agents, polyacrylamide gels, ultrafiltration devices). In this regard, see, for example, the following article, the contents of which are fully incorporated herein by reference:

[0072] -Rafael Antonio Salinas Domínguez,Miguel Domínguez Jiménez,Abdú Díaz:“Antibody Immobilization in Zinc Oxide Thin Films as an Easy-Handle Strategy for Escherichia coli Detection”-ACS Omega 2020,5,20473-20480;

[0073] -Anke K.Trilling,Jules Beekwildera,Han Zuilhof:“Antibody orientationon biosensor surfaces:a minireview”-Analyst,2013,138,1619;

[0074] -Lu Zhang,Yacine Mazouzi,Michèle Salmain,Bo Liedberg,Souhir Boujday:“Antibody-Gold Nanoparticle Bioconjugates for Biosensors:Synthesis,Characterization and Selected Applications”-Biosensors and Bioelectronics 165(2020)112370;

[0075] -Asta Makaraviciute,Carolyn D.Jackson,Paul A.Millner,AlmiraRamanaviciene:“Considerations in producing preferentially reduced half-antibody fragments”-Journal of Immunological Methods 429(2016)50-56;

[0076] -Monalisa Pal,Sanghee Lee,Donghoon Kwon,Jeongin Hwang,Hyeonjeong Lee,Seokyung Hwang,Sangmin Jeon:“Direct immobilization of antibodies on Zn-dopedFe3O4nanoclusters for detection of pathogenic bacteria”-Analytica ChimicaActa 952(2017)81-87;

[0077] -Md.Morsaline Billah,Christopher S.Hodges,Henry C.W.Hays,P.A.Millner:“Directed immobilization of reduced antibody fragments onto a novel SAM ongold for myoglobin impedance immunosensing.”-Bioelectrochemistry 80(2010)49-54;

[0078] -Hongcheng Liu&Kimberly May(2012):'Disulfide bond structures of IgGmolecules'-mAbs,4:1,17-23;

[0079] -Roberto Reverberi,Lorenzo Reverberi:“Factors affecting the antigen-antibody reaction”-Blood Transfus 2007;5:227-240;

[0080] -Harsh Sharma,Raj Mutharasan:“Half Antibody Fragments ImproveBiosensor Sensitivity without Loss of Selectivity”-Anal.Chem.2013,85,2472-2477;

[0081] -Woochang Lee,Byung-Keun Oh,Won Hong Lee,Jeong-Woo Choi:“Immobilization of antibody fragment for immunosensor application based onsurface plasmon resonance.”-Colloids and Surfaces B:Biointerfaces 40(2005)143-148;

[0082] -Yuanli Song,Hui Cai,Zhijun Tan,Nesredin Mussa,Zheng-Jian Li:“Mechanistic insights into inter-chain disulfide bond reduction of IgG1 andIgG4 antibodies”-Applied Microbiology and Biotechnology(2022)106:1057-1066;

[0083] -Nicholas G.Welch,Judith A.Scoble,Benjamin W.Muir,et al:“Orientationand characterization of immobilized antibodies for improved immunoassays”-Biointerphases 12,02D301(2017);

[0084] -Arkady A.Karyakin,Galina V.Presnova,Maya Yu.Rubtsova,AlexeyM.Egorov:“Oriented Immobilization of Antibodies onto the Gold Surfaces viaTheir Native Thiol Groups”-Anal.Chem.2000,72,3805-3811;

[0085] -Nur Mustafaoglu, Tanyel Kiziltepe, Basar Bilgicer: "Site-SpecificConjugation of Antibody on Gold Nanoparticle Surface for One-Step Diagnosis of Prostate Specific Antigen with Dynamic Light Scattering" -Nanoscale.2017June 29;9(25):8684-8694;

[0086] - Harry W Schroeder Jr, Lisa Cavacini: "Structure and Function of Immunoglobulins" - Allergy Clin Immunol. 2010 February; 125(2 0 2): S41-S52.

[0087] In case the receptor layer 5 comprises antibodies or other proteins, the aforementioned washing can be accomplished, for example, by using PBS (phosphate buffered saline), by immersion and possibly by subsequent centrifugation of the sensor.

[0088] It should be noted that according to one possible embodiment, the receptor layer 5 is capable of selectively binding to a plurality of different target analytes, for example in different regions of the receptor layer 5, and is configured such that the resulting diffraction pattern is different depending on the bound target analyte. For example, the receptor layer 5 may include different antibodies positioned in such a way that the receptor layer 5 is capable of selectively binding to different target antigens.

[0089] Although the diffraction images produced by the sensor are visible to the naked eye, they can be usefully detected by automated or partially automated systems. As an example, Figure 5 A possible system for sensing a target analyte in a sample is shown in FIG, which comprises a laser source 100, a screen 200 (preferably a satin surface) and a visual system capable of acquiring the diffraction image generated by the sensor 1, such as a camera 300 directed to the screen 200, which is connected to a control unit such as a computer 400. The latter control unit may perform a grayscale conversion of the diffraction image. Such a system may be portable, for example to perform on-site analysis. In such a case, a power supply and / or battery may be provided for the above-mentioned device.

[0090] According to a further aspect of the invention, a method for sensing a target analyte in a sample comprises the following steps:

[0091] - providing a diffraction sensor 1 according to the above-described embodiment;

[0092] - applying the sample to the receptor layer 5 of the diffraction sensor 1;

[0093] - impinging the diffraction sensor 1 with a laser beam, for example emitted by the source 100, so that the diffraction sensor 1 generates a diffraction image, for example visible to the naked eye, on the screen 200;

[0094] - comparing the diffraction image produced by the diffraction sensor 1 with a reference diffraction image obtained by hitting the diffraction sensor with an equal laser beam in the absence of the target analyte on the receptor layer 5. This comparison can be performed by a human operator or, preferably, by an automated control unit such as a computer 400;

[0095] If the diffraction image produced by the diffraction sensor 1 is different from the reference diffraction image, the presence of the target analyte in the sample is determined.

[0096] In an embodiment, the method comprises the step of converting the diffraction image into grayscale to remove the coloration that depends on the wavelength of the laser beam. This step can be performed, for example, by a control unit.

[0097] In the case where the receptor layer is capable of selectively binding to multiple target analytes, the method may also include the additional step of comparing the diffraction image generated by the sensor with a plurality of stored diffraction images (each stored diffraction image corresponding to a specific target analyte), and determining the presence of the specific 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 the specific target analyte.

[0098] In an embodiment, after the step of applying the sample to the receptor layer 5 and before the step of hitting the diffraction sensor 1 with the laser beam, the method includes an additional step of washing the diffraction sensor 1 to remove substances or molecules other than the target analyte or contaminants that may interfere with the final diffraction image (even if only as background noise) from the receptor layer 5. Such a washing step can be performed, for example, by using a buffer solution (e.g., PBS), an ionic or non-ionic detergent, or by a mild surfactant according to an appropriate process and timing.

[0099] The diffraction sensor, system and method according to the present invention may find application in various fields, some non-limiting examples of which are given below:

[0100] - Diagnosis in a medical or veterinary setting:

[0101] o Diagnosis of bacterial (such as Lyme disease, brucellosis, syphilis), viral (such as HIV, hepatitis A, B, C), and fungal (such as pathogenic yeast infections) infections by identification of the pathogen or its components;

[0102] ○ Virus testing (such as coronavirus, HIV, hepatitis, Ebola, norovirus, influenza, West Nile virus, Zika virus);

[0103] ○ Diagnosis of autoimmune diseases by demonstrating specific clinically validated markers, which are caused by the immune system abnormally producing autoantibodies that destroy cells in the body (e.g., autoantibodies that destroy insulin-producing cells in the pancreas in type 1 diabetes);

[0104] ○ Detection and quantification of hormones (e.g., human chorionic gonadotropin (hCG), follicle-stimulating hormone (FSH), testosterone);

[0105] o Screening of donated blood (e.g., to detect viral infections such as HIV);

[0106] ○Detection of drugs (e.g. amphetamine);

[0107] ○ Detection of tumor markers (e.g., prostate-specific antigen (PSA) for prostate cancer diagnosis);

[0108] ○ Diagnosis in the veterinary field (e.g., detection of African swine fever, avian influenza, bovine parvovirus, canine adenovirus, coronavirus, equine infectious anemia, feline leukemia, etc.).

[0109] -DNA search;

[0110] -Non-diagnostic applications:

[0111] ○Biosafety:

[0112] □ Epidemiological monitoring;

[0113] □ Biological weapons / bioterrorism testing;

[0114] □Testing in cosmetics (e.g. metal contamination testing);

[0115] ○Detection of pollutants:

[0116] □ Detection and screening of contaminants in food (e.g. Salmonella sp., Escherichia coli, Campylobacter sp., Staphylococcus aureus);

[0117] □ Detection and screening of environmental pollutants (e.g., detection of heavy metals in water or soil);

[0118] □Pesticide testing;

[0119] □ Gunpowder detection (eg nitrate detection by specific enzymes such as nitrate reductase).

[0120] It should be noted that in the present description and the appended claims, the term "overlapping" referring to the layers of the sensor 1 is not intended to nor necessarily implies direct contact between the mentioned overlapping layers. Such layers may thus be in direct contact with each other, or alternatively, they may have one or more intermediate layers arranged between them without impairing their overlap.

[0121] It should also be noted that for the purposes of this specification and the appended claims, unless otherwise indicated, all numbers expressing quantities, measurements, percentages, and the like are in all instances deemed to be modified by the term "about." Furthermore, all ranges include any combination of the maximum and minimum points disclosed, and include any intermediate ranges therein, which may or may not be specifically enumerated.

[0122] According to at least one of the above aspects, the present disclosure may be implemented according to one or more of the described embodiments, which are optionally combined with each other.

[0123] For the purposes of this specification and the appended claims, the terms "a" or "an" should be understood to include one or at least one and the singular includes the plural unless it is obvious that it is intended otherwise.

[0124] With respect to the description of the diffraction sensor and the system and method for sensing the target analyte in the sample given above, however, in order to meet specific needs, those skilled in the art may make many additions, modifications or replace the elements with functionally equivalent elements without departing from the scope of the appended claims.

Claims

1. A diffraction sensor (1) for sensing a target analyte, comprising: - a diffractive layer (3) comprising a plurality of surface areas (40) equal to one another and having a maximum dimension comprised between 5 μm and 50 μm, each surface area (40) comprising a diffraction grating (30) provided with grooves having a depth less than 200 nm, wherein in each of said surface areas (40) equal to one another, said diffraction grating (30) has the same conformation; - a receptor layer (5), overlapping the diffractive layer (3), configured to selectively bind to the target analyte.

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

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

4. The diffraction sensor (1) according to any one of the preceding claims, wherein the surface area (40) has a square shape and the largest dimension is a side of the square.

5. The diffraction sensor (1) according to any of the preceding claims, wherein the surface areas (40) are placed side by side.

6. The diffraction sensor (1) according to any of the preceding claims, wherein the surface areas (40) partially overlap.

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

8. The diffraction sensor (1) according to any one of the preceding claims, wherein the diffraction layer (3) comprises: - a polymer film having a thickness comprised between 5 μm and 500 μm, said polymer film being chosen from the group consisting of: polycarbonate, polyethylene terephthalate, polyvinyl chloride, polypropylene; or - an amorphous or crystalline material having a thickness comprised between 10 μm and 500 μm; or -Fiberglass material.

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

10. The diffraction sensor (1) according to the preceding claim, wherein the protective layer (4) is made of: - sulphur; or - an oxide selected from the group consisting of titanium oxide, zinc oxide, zirconium oxide, silicon oxide; or - a metal selected from the group consisting of: gold, silver, nickel, zinc, aluminum, copper.

11. The 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 the preceding claim, wherein the nanoparticles of the protective layer (4) have a size comprised between 4 nm and 30 nm, preferably less than 20 nm.

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

14. The 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 antibodies suitable for binding to the specific antigen.

15. The diffraction sensor (1) according to the preceding claim, wherein the antibody comprises half of the fraction Fab' and the fraction Fc of the antibody, wherein the disulfide bonds -SS are reduced to reduced disulfide bonds -SH suitable for binding to the protective layer (4).

16. The diffraction sensor (1) according to any one of the preceding claims, further comprising a transparent or translucent support layer (2), wherein the diffraction layer (3) overlaps the support layer (2).

17. A system for detecting a target analyte, comprising: - A diffraction sensor (1) according to any one of the preceding claims; a source (100) of a laser beam having a wavelength comprised in the visible spectrum, said source being arranged such that said diffraction sensor (1) is struck by said laser beam and generates a diffraction image; - a screen (200) placed at a distance from the diffraction sensor (1) and arranged such that the diffraction image generated by the diffraction sensor is projected thereon.

18. The system according to claim 17, wherein the laser light source (100) is arranged behind the diffraction sensor (1) so that the laser beam hits the sensor at 90° and the diffraction image is generated by the laser beam passing through the diffraction sensor (1).

19. The system according to claim 17, wherein the laser light source (100) is arranged in front of the sensor (1) so that the laser beam hits the diffraction sensor (1) diagonally and generates the diffraction image by reflecting 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 according to the preceding claim further comprises a control unit (400) which is operably connected to the visual system (300) and is configured to compare the diffraction image projected on the screen (200) with a reference diffraction image, and to determine the presence of the target analyte on the receptor layer (5) of the diffraction sensor (1) if the detected diffraction image is different from the reference diffraction image.

22. A method for detecting a target analyte in a sample, comprising the following steps: - Providing a diffraction sensor (1) according to any one of claims 1 to 17; - applying the sample to the receptor layer (5) of the diffraction sensor (1); - impinging the diffraction sensor (1) with a laser beam having a wavelength included in the visible spectrum, so that the diffraction sensor (1) generates 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 hitting the diffraction sensor (1) with the same laser beam in the absence of the target analyte on the receptor layer (5); - if the diffraction image generated by the diffraction sensor (1) is different from the reference diffraction image, the presence of the target analyte in the sample is determined.

23. The method according to the preceding claim comprises, after the step of applying the sample onto the receptor layer (5) and before the step of striking the diffraction sensor (1) with a laser beam, a step of washing the diffraction sensor (1) to remove contaminating substances or molecules or reagents different from the target analyte from the receptor layer (5).

24. The method according to claim 22 or 23, wherein the receptor layer (5) of the diffraction sensor (1) is capable of selectively binding to a plurality of target analytes, the method further comprising the steps of: - comparing the diffraction image generated by the diffraction sensor (1) with a plurality of stored diffraction images, each stored diffraction image corresponding to a specific target analyte of the plurality of target analytes; - determining the presence of a specific target analyte from the plurality of target analytes if the diffraction image generated by the diffraction sensor is consistent with a stored diffraction image for the specific target analyte.

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