A two-step method for fabricating plasmonic nanostructures for plasmonic biosensing

Abstract

The present invention relates generally to the field of biosensors, and in particular, the present invention provides a two-step method that can be applied to any one- or two-dimensional nanocrystal template for metaplasmonic biosensing to enhance surface sensitivity to biorecognition events of less than 100 nm, preferably 10-15 nm.

Description

[Technical Field]

[0001] The present invention relates generally to the field of biosensors, and in particular, the present invention provides a two-step method that can be applied to any one- or two-dimensional nanocrystal template for metaplasmonic biosensing to enhance surface sensitivity to biorecognition events below 50 nm, preferably 10-15 nm. [Background technology]

[0002] The global biosensor market was valued at US$22.4 billion in 2020 and is expected to expand at a compound annual growth rate (CAGR) of 7.9% from 2021 to 2028. The optical segment is expected to exhibit the highest CAGR of 8.6% from 2021 to 2028 (grandviewresearch.com). Among various biosensing technologies, optical biosensors based on nanoscale plasmonic phenomena have been the focus of extensive research and development in recent years due to their enhanced surface sensitivity, well-known biofunctionalization strategies, and potential for real-time, direct, label-free, and multiplexed biodetection for the development of point-of-care devices. However, the main challenges in nanoplasmonic biosensors are the high-throughput fabrication process for large-scale production and the effective optimization of the evanescent decay length for enhanced surface sensitivity in response to biorecognition events (i.e., proteins / antibodies, exosomes / antibodies, etc.). The latter has rarely been reported in the literature (Non-Patent Documents 1 and 2).

[0003] Various strategies have been proposed to enhance the surface sensitivity of nanoplasmonic biosensors for low-dimensional biorecognition events. The first strategy involves either using colloidal nanoparticles immobilized on the sensor surface to shorten the evanescent decay length and thereby improve surface sensitivity (Non-Patent Document 3), or colloidal nanoparticles decorated with secondary antibodies to form a "sandwich" assay and amplify the biosensing signal (Non-Patent Document 4). The first strategy complicates the fabrication process and has limited batch-to-batch reproducibility, while the second strategy increases the cost of the bioassay and is an indirect biosensing assay. The latter approach, involving nanoporous gold, significantly enhanced biosensing performance and surface sensitivity by up to an order of magnitude (Non-Patent Document 5).

[0004] However, the fabrication process typically involves chemical etching steps that require precise control for reproducibility in laboratory conditions. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Lopez-Munoz, 2017 [Non-patent document 2] Lopez-Munoz, 2021 [Non-patent document 3] Spackova, 2019 [Non-patent document 4] Belushkin, 2020 [Non-patent document 5] Garoli, 2019 Summary of the Invention

[0006] In the present invention, we provide herein a high-throughput technology that has remarkable potential for the mass production of direct, label-free, multiplexed biosensors with excellent surface sensitivity for thin biolayers of less than 50 nm (i.e., for protein / antibody biorecognition events, the biolayer thickness is 10 nm-15 nm). [Brief explanation of the drawings]

[0007] [Figure 1] FDTD model and electric field distribution. Morphological model of a) nanotemplate covered with 70 nm flat gold film and b) 60 nm flat gold film + 10 nm high gold nanoroughness layer. Simulation of electric field distribution of c) 70 nm flat gold film and d) 60 nm flat gold film + 10 nm high gold nanoroughness layer. [Figure 2] Figure 1 shows FDTD results for bulk and surface sensitivity. a) Plasmon band shift under high medium refractive index change (Δn = 0.2 RIU) for flat (70 nm Au film) and Flat+GLAD (60 nm flat Au film + 10 nm high Au nano-roughness layer). b) Plasmon band shift for 10 nm and 15 nm biolayers (n = 1.45 RIU) to mimic the interaction between small and large proteins in protein / antibody biorecognition events. c) FDTD simulation results for bulk and surface sensitivity, showing a decrease in bulk sensitivity and a more than 2.5-fold enhancement in surface sensitivity, resulting in enhanced biosensing performance for protein / antibody biorecognition events up to a height of 15 nm. [Figure 3] Figure 1 shows the experimental setup: a) Schematic of the experimental optical setup; b) Photograph of the experimental optical setup, with the inset showing the integrated plasmonic sensor with two sensing areas. [Figure 4]Figure 1 shows the bulk and surface sensitivity with and without nanoroughness under two different GLAD angle deposition conditions. a) Plasmon band shifts under different refractive index conditions for a Flat+GLAD (60 nm flat Au film + 5 nm high Au nanoroughness) sensor. b) Bulk sensitivity estimation for a 70 nm flat control (0°) and 60 nm + 5 nm nanoroughness with GLAD angles of 82° and 86°. c) Plasmon band shifts after deposition of a 15 nm aluminum oxide layer (n = 1.45 RIU) to mimic the maximum estimated height of antigen / antibody biorecognition events, considering the antibody height of around 6 nm to 7 nm and the target protein diameter of up to 6 nm to 8 nm. d) Experimental results of bulk and surface sensitivity. The bulk sensitivity is reduced and the surface sensitivity is enhanced by nearly 1.5 times, resulting in enhanced biosensing performance for protein / antibody biorecognition events up to a height of 15 nm. [Figure 5] SEM images of various deposition conditions: a) control (flat 70 nm Au), b) 60 nm flat + 5 nm Au nanoroughness at 82°, and c) 60 nm flat + 5 nm high Au nanoroughness at 86°. 82° exhibits higher roughness relative to the control surface, while 86° exhibits lower roughness relative to the 82° condition, with a noticeable shadowing effect. [Figure 6] Experimental results using nanoroughness GLAD layers of different heights. a) Plasmonic response of metasurfaces with a 70 nm flat Au film (control) and a fixed 60 nm flat Au film + nanoroughness layers of different heights ranging from 5 nm to 15 nm under a GLAD deposition angle of 82°. b) Increase in surface sensitivity with nanoroughness GLAD layers of different heights after deposition of 15 nm alumina to mimic a biological layer, with the largest biosensing improvement of up to 2.3 times for the 15 nm nanoroughness. [Figure 7] Figure 1 shows the FDTD simulation results for estimating the optimal gold layer thickness. Various simulations of the reflectance spectra of Blu-ray disc-based one-dimensional nanocrystals coated with different gold layer thicknesses. The figure shows that an optimal gold layer thickness of 70 nm is achieved with the minimum reflectance value closest to 0 a.u. [Figure 8]This figure shows the scheme of the GLAD method: (a) Diagram of the main process; (b) Film deposition process on a substrate with shadowing effect [Sensors 2022, 22, 651. https: / / doi.org / 10.3390 / s22020651]. [Figure 9] A diagram showing the GLAD instrument scheme: (a) deposition chamber, (b) GLAD manipulator, (c) magnetron with metal target, (d) vacuum pump, M1 DC motor for rotation (φ), M2 stepper for angular change (α) [Sensors 2022, 22, 651. https: / / doi.org / 10.3390 / s22020651]. [Figure 10] Figure 1 shows the evaporation flux at different angles. The left figure shows the evaporation flux at normal incidence. The right figure shows the evaporation flux at incidence angles α and -α. DETAILED DESCRIPTION OF THE INVENTION

[0008] definition As used herein, "FDTD" shall be understood to refer to the Finite-Difference Time-Domain method used to solve Maxwell's equations in modeled nanoscale optical elements.

[0009] As used herein, "plasmon band" shall be understood as the range or region of wavelengths over which a metal nanomaterial absorbs light.

[0010] As used herein, "metaplasmonic" is to be understood as a plasmonic effect based on highly ordered and quasi-ordered metallic nanostructures.

[0011] As used herein, "metaplasmonic biosensing" shall be understood as biosensing based on highly ordered and quasi-ordered metallic nanostructures.

[0012] As used herein, "nanocrystal" shall be understood as a crystalline structure having at least one dimension less than 100 nanometers.

[0013] As used herein, "minimum reflectivity value" shall be understood as the reflectance value closest to 0 in the reflectance spectrum that refers to the maximum light absorption of the material.

[0014] As used herein, "nano-roughness" shall be understood as an uneven or irregular surface having at least one dimension less than 100 nanometers.

[0015] As used herein, "GLAD" shall be understood to mean Glancing angle deposition, a variation of oblique deposition with biaxial rotation.

[0016] As used herein, "evaporation flux" shall be understood as the magnitude and direction of evaporation of a material.

[0017] As used herein, "plasmonic nanostructures" shall be understood to be noble metal-based structures having at least one dimension less than 100 nanometers.

[0018] As used herein, "normal incidence of evaporation flux" shall be understood as the perpendicular incidence between the substrate on which the material is deposited and the direction of evaporation of the material.

[0019] As used herein, a "biorecognition event" is to be understood as a biochemical interaction between a bioreceptor, i.e., an antibody, and a target, i.e., a protein that has a specific affinity for the selected antibody.

[0020] As used herein, "optimum plasmon coupling" is to be understood as the condition where the minimum reflectivity value (maximum absorption) in the plasmon band is achieved closest to the 0 a.u. value. This involves the optimum metal layer thickness for achieving the minimum reflectivity value (maximum absorption) for the plasmon band. As shown in Figure 7, the optimum thickness for gold is 70 nm, below and above which the plasmon coupling is not optimal.

[0021] Detailed Description of the Invention This invention provides a two-step method that can be applied to any one- or two-dimensional nanocrystal template for metaplasmonic biosensing to enhance surface sensitivity for biorecognition events of less than 100 nm, preferably less than 50 nm, and more preferably 10-15 nm, corresponding to typical protein / antibody biorecognition events (antibody heights of approximately 6-7 nm and target protein diameters of up to 6-8 nm) by generating metal nanoparticles on the surface of a metal layer. FDTD optical simulations were performed based on a model of a one-dimensional nanocrystal template for a Blu-ray disc coated with a 70 nm Au layer, corresponding to the optimal thickness for achieving the minimum reflectivity value in the plasmon band, and a one-dimensional nanocrystal template coated in the first step with a 60 nm Au layer (below the optimal thickness for achieving the minimum reflectivity value in the plasmon band) and then coated with a 10 nm-high random nanoroughness layer (Figure 1). Optical simulations demonstrate that, compared to a flat Au layer, this additional layer localizes the electromagnetic field near the surface, reducing the evanescent field decay length and creating an optimal environment for biosensing thin biolayers (Figures 1d and 2a). Second, this additional layer not only enhances the sensor's surface sensitivity by nearly 2.5 times when simulating 10- and 15-nm biolayers (Figures 2b and 2c), but also increases the optical absorption (or minimum reflectivity value in the plasmon band) due to increased nanoroughness-induced scattering. In other words, this nanoroughness promotes higher energy transfer between the metaplasmonic substrate and light via light scattering (evident by a wider and deeper plasmon band).

[0022] Experimentally, we optically characterized one-dimensional plasmonic nanostructures using the experimental setup shown in Figure 3. As a first step, we evaluated angle deposition (GLAD) (the technique is shown in Figure 8). Three conditions were tested (using the deposition system shown in Figure 9): a 70 nm Au flat layer using a GLAD angle of 0° (control), a 60 nm Au layer at 0° + a 5 nm high nanorough layer at 82°, and a 60 nm Au layer at 0° + a 5 nm high nanorough layer at 86°. Figure 4b shows that the addition of the nanorough layer reduces the bulk sensitivity of the plasmonic metasurface in both angle conditions. These results are in good agreement with theoretical results. The plasmonic metasurface was then coated with a 15 nm alumina layer by electron beam evaporation to mimic a biological layer, and the plasmon band shift was measured. As shown in Figure 4c and summarized in Figure 4d, the nanoroughness layer increased the biosensing performance by up to 1.5 times, demonstrating that the presence of this second nanoroughness layer has a significant impact on biosensing performance. As can be seen in Figure 5, there was a clear increase in metasurface roughness after the additional GLAD, which was more pronounced at 82° than at 86° as a result of the higher shadowing effect at the higher GLAD incidence angles.

[0023] Finally, different nano-roughness layer heights were evaluated: 60 nm flat layer GLAD 0° + 5 nm GLAD 82°, 60 nm flat layer GLAD 0° + 10 nm GLAD 82°, and 60 nm flat layer GLAD 0° + 15 nm GLAD 82°. As can be seen in Figure 6, the minimum reflectivity value decreases with nano-roughness layer height, and the surface sensitivity increases with height, with a maximum enhancement of 2.3 times observed in the +15 nm GLAD 82° condition.

[0024] This method achieved up to a 2.3-fold / 2.5-fold enhancement in biosensing performance compared to flat one- or two-dimensional plasmonic metasurfaces. Indeed, the addition of this novel GLAD layer enhances surface sensitivity, plasmonic coupling, and consequently biosensing performance by creating gold nanoparticles / nanoroughness on the gold layer surface. Finally, the presence of these gold nanoparticles / nanoroughness under appropriate plasmonic coupling conditions enhances surface sensitivity by increasing the number of "hot spots" and plasmonic coupling for any one- or two-dimensional periodic plasmonic nanostructure (i.e., nanoholes, nanolattices, nanopillars, etc.). The increased number of hot spots localizes the evanescent field decay length, making it suitable for detecting low-dimensional biomarkers (i.e., insulin) that are extremely difficult to detect using conventional plasmonic sensors.

[0025] Thus, in a first aspect, the present invention refers to a plasmonic nanostructure biosensor comprising a substrate and a metal film disposed on the substrate, wherein the metal film is disposed on the substrate under two deposition conditions: a first layer is deposited at a thickness below the optimum plasmonic coupling, and a second layer is deposited at a fixed deposition angle using oblique evaporation.

[0026] Preferably, the first layer is deposited under an evaporative flux, preferably at an angle of incidence of about 0° to about 180°, preferably 45° to 135°, preferably 70° to 110°, preferably 80° to 100°, more preferably 85° to 95°, and even more preferably at normal incidence of the evaporative flux, see FIG.

[0027] In preferred embodiments of the first aspect of the present invention, the substrate comprises or is made from silicon, silicon dioxide, silicon nitride, glass, diamond, quartz, magnesium fluoride (MgF2), calcium fluoride (CaF2), ZnSe, germanium, or a polymer. Preferably, the substrate is a crystal or nanocrystal, more preferably a one- or two-dimensional nanocrystalline template (i.e., a one-dimensional nanocrystal of a Blu-ray disc).

[0028] In another preferred embodiment of the first aspect of the present invention, the metal is a noble metal, a transition metal, or an alkali metal. Highly preferably, the metal comprises or is gold. In any case, it is noted that the metal used in the nanoplasmonic structures described herein is selected based on its surface plasmon properties when the surface is illuminated with an incident light source. Thus, the metal used can be a noble metal or any metal selected from the group consisting of gold, rhodium, palladium, silver, osmium, iridium, platinum, titanium, and aluminum.

[0029] It is further noted that, in the context of the present invention, preferably, when gold is used as the metal, a thickness below optimal plasmon coupling under normal incidence of the evaporation flux is understood to be less than 70 nm. Thus, in another preferred embodiment of the first aspect of the present invention, the metal is preferably gold, the first layer has a thickness of less than 70 nm, the second layer has a thickness in the range of 1 nm to 40 nm, and the total thickness of the first and second layers combined is 65 nm to 100 nm, preferably in the range of 65 nm to 80 nm, more preferably about 70 nm. It is noted that for all of these ranges, the specification expressly encompasses all specific values ​​within the range, including the lower and upper limits of the range. That is, when the thickness of a second layer is stated to be in the range of 1 nm to 40 nm, it should be understood to explicitly encompass any value between 1 nm and 40 nm, particularly 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, and 40 nm. The same is true for any of the other ranges given throughout this specification. Additionally, any combination of values ​​within the ranges set forth herein for the first layer and second layer is considered or understood to be encompassed herein so long as it provides or results in a total thickness of the first layer and second layer combined within the specified ranges.

[0030] In another preferred embodiment of the first aspect of the present invention, the metal is preferably gold, the first layer has a thickness of 40 nm to 65 nm, preferably 50 nm to 65 nm, more preferably about 60 nm, the second layer has a thickness in the range of 1 nm to 30 nm, preferably 1 nm to 20 nm, more preferably 1 nm to 15 nm, and the total thickness of the first and second layers combined is 65 nm to 100 nm, preferably in the range of 65 nm to 80 nm, more preferably about 70 nm.

[0031] In another preferred embodiment of the first aspect of the present invention, the metal disposed on the substrate is gold, the first layer has a thickness of 40 nm to 65 nm, preferably 50 nm to 65 nm, and more preferably about 60 nm, the second layer has a thickness of 1 nm to 30 nm, preferably 1 nm to 20 nm, and more preferably in the range of 1 nm to 15 nm, and even more preferably 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, or 15 nm, and the total thickness of the first and second layers combined is in the range of 65 nm to 80 nm, and more preferably about 70 nm.

[0032] In another preferred embodiment of the first aspect of the present invention, the metal film is further characterized by comprising a plurality of nanoelements, e.g., capture agents, arranged in a predetermined pattern on the metal film, each of the nanoelements having a dimension of less than 100 nm, preferably less than 50 nm, preferably between 1 nm and 15 nm, more preferably between 5 nm and 10 nm.

[0033] The nanoelements can be arranged in any pattern that produces the desired optical properties of the nanoplasmonic biosensor, including both periodic and aperiodic patterns, e.g., pseudorandom and random patterns. Thus, in some embodiments of this aspect, the pattern of nanoelements is a periodic pattern. In some embodiments of this aspect, the pattern of nanoelements is a non-periodic pattern, e.g., a pseudorandom pattern or a random pattern.

[0034] In some embodiments, it may be desirable to activate the metal surface of a nanoplasmonic structure using an activating agent. As used herein, "activating" a metal surface refers to treating the metal surface with an activating agent to enable, tolerate, or enhance the binding of nanoelements, such as capture agents. The activating agent can be selected based on the nature of the nanoelements used with the nanoplasmonic structure, for example, whether the nanoelements are proteins or nucleic acids. Thus, in some embodiments, when the nanoelements are proteins, the activating agent used to activate the metal surface is piranha solution. Thus, the metal surface of a plasmonic nanostructure can be functionalized with one or more specific capture agents. As used herein, "functionalization" refers to the addition of one or more specific nanoelements to the metal surface of a plasmonic nanostructure according to the present invention. In some embodiments, the metal surface is first activated and then functionalized. In other embodiments, the functionalization of the metal surface can be performed without activation.

[0035] Nanoelements, such as capture agents, used to functionalize nanoplasmonic nanostructures according to the present invention must have specific binding properties for one or more biomolecular targets. As used herein, "capture agent" refers to any of a variety of specific binding molecules, including, but not limited to, DNA oligonucleotides, RNA oligonucleotides, peptides, proteins (e.g., transcription factors, antibodies or antibody fragments thereof, receptors, recombinant fusion proteins, or enzymes), small organic molecules, or any combination thereof, that can be immobilized on the surface of the plasmonic nanostructures described herein. In some embodiments, capture agents are immobilized in a periodic manner. For example, one or more specific immobilized capture agents can be aligned and arranged at one or more distinct locations on the surface of the plasmonic nanostructure. In some such embodiments, capture agents specific for different biomolecular targets can be immobilized at such distinct locations on the surface of the plasmonic structure, allowing the structure to be used for detecting multiple biomolecular targets in a sample. In other embodiments, capture agents are immobilized in a non-periodic or random manner.

[0036] Such functionalized biosensors are useful for detecting biomolecular interactions, including, but not limited to, DNA-DNA, DNA-RNA, DNA-protein, RNA-RNA, RNA-protein, and protein-protein interactions. For example, a plasmonic nanostructure having multiple DNA oligonucleotides immobilized on its surface can be used to detect the presence of proteins, such as transcription factors, that bind to one or more of the oligonucleotides and are present in a sample in contact with the substrate layer.

[0037] Thus, in some embodiments, the metal surface of a plasmonic nanostructure described herein is functionalized with a capture agent comprising one or more of a plurality of immobilized DNA oligonucleotides. In some embodiments, the metal surface of a plasmonic nanostructure described herein is functionalized with a capture agent comprising one or more of a plurality of immobilized RNA oligonucleotides. In some embodiments, the metal surface of a plasmonic nanostructure described herein is functionalized with a capture agent comprising one or more of a plurality of immobilized peptides. In some embodiments, the metal surface of a plasmonic nanostructure described herein is functionalized with a capture agent comprising one or more of a plurality of immobilized proteins. In some such embodiments, the protein is an antigen. In other such embodiments, the protein is a polyclonal antibody, a monoclonal antibody, a single-chain antibody (scFv), a F(ab) fragment, a F(ab')2 fragment, or an Fv fragment. In other such embodiments, the protein is an enzyme, a transcription factor, a receptor, or a recombinant fusion protein.

[0038] In a second aspect of the present invention, there is provided a method of manufacturing a plasmonic nanostructure biosensor, the method comprising: i. a first layer of a metal film is deposited on a substrate at a thickness below optimal plasmon coupling; ii. the second layer is deposited using oblique deposition at a fixed deposition angle; The present invention relates to a method that includes disposing a metal film on a substrate under two deposition conditions:

[0039] Preferably, the first layer is deposited under an evaporative flux at an incidence angle of 70° to 110°, preferably 80° to 100°, more preferably 85° to 95°, and even more preferably under normal incidence of the evaporative flux.

[0040] In preferred embodiments of the second aspect of the present invention, the substrate comprises or is made from silicon, silicon dioxide, silicon nitride, glass, diamond, quartz, magnesium fluoride (MgF2), calcium fluoride (CaF2), ZnSe, germanium or a polymer, and preferably the substrate is crystalline or nanocrystalline.

[0041] In another preferred embodiment of the second aspect of the present invention, the metal film comprises or is made from a noble metal, a transition metal or an alkali metal, or any combination thereof, preferably the metal is gold.

[0042] In another preferred embodiment of the second aspect of the present invention, the substrate is a one- or two-dimensional nanocrystal template (ie, a one-dimensional nanocrystal of a Blu-ray disc).

[0043] In another preferred embodiment of the second aspect of the present invention, the second layer is deposited using glancing angle evaporation.

[0044] In another preferred embodiment of the second aspect of the present invention, the metal disposed on the substrate is gold, the first layer is deposited to a thickness of less than 70 nm, and the second layer is deposited to a thickness in the range of 1 nm to 40 nm. Preferably, the metal disposed on the substrate is gold, the first layer is deposited to a thickness of 40 nm to 65 nm, preferably 50 nm to 65 nm, more preferably about 60 nm, and the second layer is deposited to a thickness in the range of 1 nm to 30 nm, preferably 1 nm to 20 nm, more preferably 1 nm to 15 nm. More preferably, the metal disposed on the substrate is gold, and the first and second layers are deposited to a total thickness in the range of 65 nm to 100 nm, preferably 65 nm to 80 nm. Even more preferably, the total thickness of the first and second layers combined is about 70 nm.

[0045] In another preferred embodiment of the second aspect of the present invention, the metal disposed on the substrate is gold and the second layer is deposited using glancing angle evaporation, the first layer being deposited to a thickness of 40 nm to 65 nm, preferably 50 nm to 65 nm, more preferably about 60 nm, and the second layer being deposited to a thickness in the range of 1 nm to 30 nm, preferably 1 nm to 20 nm, more preferably 1 nm to 15 nm, with the total thickness of the first and second layers combined being in the range of 65 nm to 80 nm, preferably about 70 nm.

[0046] In another preferred embodiment of the second aspect of the present invention, the second layer is preferably deposited using glancing angle deposition at a fixed deposition angle of less than 90°, preferably between 70° and 90°, more preferably between 80° and 90°, preferably about 82° or about 86°.

[0047] In another preferred embodiment of the second aspect of the present invention, the metal film is further functionalized to obtain a plurality of nanoelements arranged in a predetermined pattern on the metal film, said nanoelements each having a dimension of less than 100 nm, preferably between 1 nm and 15 nm, more preferably between 5 nm and 10 nm. It is noted that such nanoelements are preferably capture elements as defined and designated in the first aspect of the present invention.

[0048] In a third aspect, the present invention refers to a plasmonic nanostructure biosensor obtained according to the method of the second aspect of the present invention or according to any of its preferred embodiments.

[0049] A fourth aspect of the present invention refers to the in vitro use of a plasmonic nanostructure according to the first aspect of the invention or any of its preferred embodiments, or a plasmonic nanostructure according to the third aspect of the invention, for detecting one or more biomolecular targets, wherein the biorecognition event is less than 100 nm, preferably between 10 nm and 15 nm.

[0050] In a fifth aspect of the present invention there is provided a plasmonic nanostructure biosensor system for detecting one or more biomolecular targets, preferably comprising: a. a plasmonic nanostructure biosensor as defined in the first aspect of the present invention or any of its preferred embodiments or as defined in the third aspect of the present invention, comprising a substrate and a metal film disposed on the substrate, wherein the metal film comprises a plurality of nanoelements arranged in a predetermined pattern, each of the nanoelements having a dimension of less than 100 nm, preferably between 1 nm and 15 nm, more preferably between 5 nm and 10 nm; b. an apparatus for contacting one or more samples containing one or more biomolecular targets with the metal film surface(s) of the plasmonic nanostructure biosensor; c. an incident light source for irradiating the surface of the metal film to generate surface plasmons; d. an optical detection system for collecting and measuring light displaced from the illuminated metal film, the displaced light being indicative of surface plasmon resonance on one or more surfaces of the metal film; and The present invention refers to a plasmonic nanostructure biosensor system comprising:

[0051] In a preferred embodiment of the fifth aspect of the present invention, the device for contacting one or more samples comprises a fluidic system.

[0052] In a sixth aspect of the present invention there is provided a method for detecting one or more biomolecular targets, comprising the steps of: a. iii. A plasmonic nanostructure biosensor as defined in the first aspect of the present invention or any of its preferred embodiments or as defined in the third aspect of the present invention, comprising a substrate and a metal film disposed on the substrate, wherein said metal film comprises a plurality of nanoelements arranged in a predetermined pattern, each of said nanoelements having a dimension of less than 100 nm, preferably between 1 nm and 15 nm, more preferably between 5 nm and 10 nm; iv. A device for contacting one or more samples containing one or more biomolecular targets with the metal film surface(s) of the plasmonic nanostructure biosensor; v. an incident light source for irradiating the surface of the metal film to generate the surface plasmons; vi. an optical detection system for collecting and measuring light displaced from the illuminated metal film, the displaced light being indicative of surface plasmon resonance on one or more surfaces of the metal film; and providing a plasmonic nanostructure biosensor system comprising: b. contacting one or more samples containing one or more biomolecular targets with the metal film surface of the plasmonic nanostructure biosensor; c. illuminating one or more surfaces of the metal film of the plasmonic nanostructure biosensor with an incident light source to generate surface plasmons before and after contact with one or more samples; d. collecting and measuring light displaced from the illuminated film with an optical detection system before and after contact with one or more samples; e. detecting one or more biomolecular targets based on a change or difference in the measurement of light displaced from the illuminated membrane before and after contact with one or more samples; The present invention relates to a method including:

[0053] In a preferred embodiment of the sixth aspect of the present invention, the biomolecular target is a eukaryotic cell, a eukaryotic cell component, a prokaryotic cell, a prokaryotic cell component, a virus particle, a protein, an oligonucleotide, a prion, a toxin, or any combination thereof.

[0054] In another preferred embodiment of the sixth aspect of the present invention, said collected light comprises light in transmission mode, reflection mode or a combination thereof.

[0055] In another preferred embodiment of the sixth aspect of the present invention, the step of measuring the displaced light comprises measuring the light over a spectral range selected to include at least one plasmon band.

[0056] In yet another preferred embodiment of the sixth aspect of the present invention, the change in the measurement of displaced light before and after contact is a resonance peak shift, a change in resonance peak intensity, a broadening of the resonance peak, a distortion of the peak resonance or a change in refractive index.

[0057] The following examples are merely illustrative of the present invention and are not intended to limit the invention. [Example]

[0058] Materials and Methods: FDTD Simulation. Three-dimensional FDTD simulations were performed using commercially available software (FDTD solution, Lumerical, Inc., Vancouver, Canada). The structural parameters of the Blu-ray disc used in the simulations (i.e., grating period 320 nm, grating width 100 nm, and height 20 nm) were as reported for atomic force microscopy (AFM) images in previous results (Lopez et al., 2018). Periodic boundary conditions were used for the x- and y-axes, and a perfectly matched layer (PML) approach was used for the z-axis, with a uniform mesh size of 2 nm for all axes. FDTD simulations were performed in the range of 400 nm to 1000 nm under TM polarization with a variable oblique light incidence angle of 40°. The optical constants of polycarbonate and gold were taken from Sultanova et al., 2009 and Wakaki et al., 2007, respectively. The refractive index of the biological layer was fixed at n = 1.45 RIU according to a previous report (Xu, 2019).

[0059] Fabrication of plasmonic metasurfaces. Single-layer recordable Blu-ray discs (43743, Verbatim, Taiwan) were used as one-dimensional nanocrystal templates after removing the protective (thin polycarbonate) and reflective (aluminum) films. The discs were then cut into individual plasmonic chips (5.6 cm in size). 2 The reflective film was removed by cutting the polycarbonate substrate into 1 / 4" thick (0.1 mm thick) and then immersed in a hydrochloric acid solution (2M HCl) overnight. The polycarbonate substrate was rinsed with deionized water and dried with nitrogen. The film was deposited using a PVD (physical vapor deposition) system and the GLAD (glancing angle deposition) technique. The magnetron sputtering technique was applied, and an Au metal target was used. The target had a purity of 5N (99.9995%) and a diameter of 50 mm, and a 50 W DC-MF (direct current medium frequency) power supply was used. The base vacuum and operating pressure were 5 × 10, respectively. -6 mbar and 8×10 -3For the reference sample, a 70 nm thick gold layer was deposited without GLAD, while for the other samples, a first 60 nm layer was deposited without GLAD, followed by nano-roughness layers with heights ranging from 5 nm to 15 nm, at GLAD angles of 82° and 86°.

[0060] A microfluidic system for in-solution detection was developed using microfluidic multichannels patterned on a 140 μm-thick double-sided adhesive tape sheet (Mcs-foil-008, Microfluidic ChipShop GmbH, Germany). The proposed design integrates two sensing areas to enable multiplexed biodetection. A 2 mm-thick patterned polymethyl methacrylate (PMMA) lid was added as a cover to facilitate the connection of fluidic tubing.

[0061] The chip was fixed to a custom-built optical platform for reflectance measurements. It was connected to a peristaltic pump with adjustable pumping speed to ensure a constant liquid flow rate. Reflectance measurements were performed under TM-polarized light from a compact stabilized broadband light source (SLS201L, Thorlabs, Germany) at a fixed angle of 40°. The incident excitation plane was aligned perpendicular to the nanograting direction. The reflected light was collected and fiber-coupled to a compact charge-coupled device (CCD) spectrometer (Exemplar UV-NIR, BWTek, Germany). Reflectance spectra were acquired every 1 ms, and 50 consecutive spectra were measured and averaged to obtain the final spectrum. These acquisition parameters were selected to obtain an optimal signal-to-noise (S / N) ratio without significantly increasing data acquisition time. Changes in the resonant peak position (λSPR) were tracked by peak analysis using Origin 2018 software (OriginPro, OriginLab Co., Northampton, USA).

[0062] References Belushkin, Alexander, et al. "Rapid and digital detection of inflammatory biomarkers enabled by a novel portable nanoplasmonic imager." Small 16.3 (2020): 1906108. Garoli, Denis, et al. "Nanoporous gold metamaterials for high sensitivity plasmonic sensing." Nanoscale Horizons 4.5 (2019): 1153-1157. Lopez-Munoz, Gerardo A., et al. "Plasmonic nanocrystals on polycarbonate substrates for direct and label-free biodetection of Interleukin-6 in bioengineered 3D skeletal muscles." Nanophotonics 10.18 (2021): 4477-4488. Lopez-Munoz GA, Estevez MC, Vazquez-Garcia M, Berenguel-Alonso M, Alonso-Chamarro J, Homs-Corbera A, Lechuga LM. Gold / silver / gold trilayer films on nanostructured polycarbonate substrates for direct and label-free nanoplasmonic biosensing. J Biophotonics 2018,11,e201800043. https: / / doi.org / 10.1002 / jbio.201800043 Lopez, Gerardo A., et al. "Recent advances in nanoplasmonic biosensors: applications and lab-on-a-chip integration." Nanophotonics 6.1 (2017): 123-136. Spackova, Barbora, Maria Laura Ermini, and Jiri Homola. "High-performance biosensor exploiting a light guidance in sparse arrays of metal nanoparticles." Optics Letters 44.7 (2019): 1568-1571. Sultanova N, Kasarova S, Nikolov I. Dispersion properties of optical polymers. Acta Phys Pol A 2009,116,585-587. https: / / doi.org / 10.12693 / APhysPolA.116.585 Wakaki M, Kudo K, Shibuya, T.Physical properties and data of optical materials 2007. Boca Raton (Fla.) : CRC press. Xu Y, Bai P, Zhou X, Akimov Y, Png CE, Ang LK, Knoll W, Wu, L. Optical Refractive Index Sensors with Plasmonic and Photonic Structures: Promising and Inconvenient Truth. Adv Opt Mater 2019,7,31-33. https: / / doi.org / 10.1002 / adom.201801433

[0063] CLAUSES 1. A plasmonic nanostructure biosensor comprising a substrate and a metal film disposed on the substrate, wherein the metal film is disposed on the substrate under two deposition conditions: a first layer is deposited, preferably under normal incidence of the evaporation flux, at a thickness below optimal plasmon coupling, and a second layer is deposited at a fixed deposition angle using oblique evaporation.

[0064] 2. The plasmonic nanostructure biosensor of item 1, wherein the substrate comprises or is made from silicon, silicon dioxide, silicon nitride, glass, diamond, quartz, magnesium fluoride (MgF2), calcium fluoride (CaF2), ZnSe, germanium, or a polymer.

[0065] 3. The plasmonic nanostructure biosensor of any one of items 1 or 2, wherein the metal is a noble metal, a transition metal, or an alkali metal.

[0066] 4. The plasmonic nanostructure biosensor of any one of items 1 or 2, wherein the metal is gold.

[0067] 5. The plasmonic nanostructure biosensor of any one of items 1 to 4, wherein the substrate is a one-dimensional or two-dimensional nanocrystal template (i.e., a one-dimensional nanocrystal of a Blu-ray disc).

[0068] 6. The plasmonic nanostructure biosensor of any one of items 1 to 5, wherein the first layer has a thickness of less than 70 nm, the second layer has a thickness in the range of 1 nm to 40 nm, and the total thickness of the first and second layers is in the range of 65 nm to 100 nm, preferably 65 nm to 80 nm.

[0069] 7. The plasmonic nanostructure biosensor of item 6, wherein the first layer has a thickness of 40 nm to 65 nm, preferably 50 nm to 65 nm, more preferably about 60 nm, and the second layer has a thickness in the range of 1 nm to 30 nm, preferably 1 nm to 20 nm, more preferably 1 nm to 15 nm.

[0070] 8. The plasmonic nanostructure biosensor of any one of items 6 or 7, wherein the total thickness of the first layer and the second layer is in the range of 65 nm to 80 nm.

[0071] 9. The plasmonic nanostructure biosensor of item 8, wherein the total thickness of the first layer and the second layer is about 70 nm.

[0072] 10. The plasmonic nanostructure biosensor of any one of items 1 to 6, wherein the metal disposed on the substrate is gold, the first layer has a thickness of 40 nm to 65 nm, preferably 50 nm to 65 nm, and more preferably about 60 nm, the second layer has a thickness in the range of 1 nm to 30 nm, preferably 1 nm to 20 nm, and more preferably 1 nm to 15 nm, and the total thickness of the first layer and the second layer is in the range of 65 nm to 80 nm.

[0073] 11. The plasmonic nanostructure biosensor of item 10, wherein the total thickness of the first layer and the second layer is about 70 nm.

[0074] 12. The plasmonic nanostructure biosensor of any one of items 1 to 8, wherein the metal film further comprises a plurality of nanoelements arranged in a predetermined pattern on the metal film, each of the nanoelements having a dimension of less than 100 nm, preferably 1 nm to 15 nm, more preferably 5 nm to 10 nm.

[0075] 13. A method of manufacturing a plasmonic nanostructure biosensor, the plasmonic nanostructure comprising a substrate and a metal film disposed on the substrate, the method comprising: i. one first layer of a metal film is deposited on a substrate, preferably under normal incidence of the evaporation flux, with a thickness below the optimum plasmon coupling; ii. the second layer is deposited using oblique deposition at a fixed deposition angle; and disposing a metal film on a substrate under two deposition conditions:

[0076] 14. The method of item 13, wherein the substrate comprises or is made of silicon, silicon dioxide, silicon nitride, glass, diamond, quartz, magnesium fluoride (MgF2), calcium fluoride (CaF2), ZnSe, germanium, or a polymer, and preferably, the substrate is crystalline or nanocrystalline.

[0077] 15. The method of any one of items 13 or 14, wherein the metal film comprises or is made from a noble metal, a transition metal or an alkali metal, or any combination thereof, and preferably the metal is gold.

[0078] 16. The method of any one of items 13 to 15, wherein the substrate is a one-dimensional or two-dimensional nanocrystal template (i.e., a one-dimensional nanocrystal of a Blu-ray disc).

[0079] 17. The method of any one of claims 13 to 16, wherein the second layer is deposited using glancing angle deposition and the metal is gold.

[0080] 18. The method of any one of items 13 to 17, wherein the first layer has a thickness of less than 70 nm and the second layer has a thickness in the range of 1 nm to 40 nm.

[0081] 19. The method of any one of items 13 to 17, wherein the first layer has a thickness of 40 nm to 65 nm, preferably 50 nm to 65 nm, more preferably about 60 nm, and the second layer has a thickness in the range of 1 nm to 30 nm, preferably 1 nm to 20 nm, more preferably 1 nm to 15 nm.

[0082] 20. The method of any one of items 18 to 19, wherein the total thickness of the first layer and the second layer is in the range of 65 nm to 100 nm, preferably 65 nm to 80 nm.

[0083] 21. The method of any one of items 18-19, wherein the total thickness of the first layer and the second layer is about 70 nm.

[0084] 22. The method of any one of items 13 to 21, wherein the metal disposed on the substrate is gold, the second layer is deposited using glancing angle deposition, the first layer has a thickness of 40 nm to 65 nm, preferably 50 nm to 65 nm, more preferably about 60 nm, the second layer has a thickness in the range of 1 nm to 30 nm, preferably 1 nm to 20 nm, more preferably 1 nm to 15 nm, and the total thickness of the first layer and the second layer is in the range of 65 nm to 80 nm, preferably about 70 nm.

[0085] 23. The method of any one of items 13 to 22, wherein the second layer is preferably deposited using glancing angle deposition at a fixed deposition angle of less than 90°, preferably between 70° and 90°, more preferably between 80° and 90°.

[0086] 24. The method of any one of items 13 to 23, wherein the metal film further comprises a plurality of nanoelements arranged in a predetermined pattern on the metal film, each of the nanoelements having a dimension of less than 100 nm, preferably between 1 nm and 15 nm, more preferably between 5 nm and 10 nm.

[0087] 25. A plasmonic nanostructure biosensor obtained according to any one of the methods of items 13 to 24.

[0088] 26. In vitro use of a plasmonic nanostructure according to any of items 1 to 12 or a plasmonic nanostructure according to item 25 for detecting one or more biomolecular targets, preferably wherein the biorecognition event is less than 100 nm, preferably between 10 nm and 15 nm.

[0089] 27. A plasmonic nanostructure biosensor system for the detection of one or more biomolecular targets, preferably comprising: a. A plasmonic nanostructure biosensor comprising a substrate and a metal film disposed on the substrate, as defined in any one of items 1 to 12 or as defined in item 25, wherein the metal film comprises a plurality of nanoelements arranged in a predetermined pattern, each of the nanoelements having a dimension of less than 100 nm, preferably 1 nm to 15 nm, more preferably 5 nm to 10 nm; b. an apparatus for contacting one or more samples containing one or more biomolecular targets with the metal film surface(s) of the plasmonic nanostructure biosensor; c. an incident light source for irradiating the surface of the metal film to generate surface plasmons; d. an optical detection system for collecting and measuring light displaced from the illuminated metal film, the displaced light being indicative of surface plasmon resonance on one or more surfaces of the metal film; and A plasmonic nanostructure biosensor system comprising:

[0090] 28. The plasmonic nanostructure biosensor system of item 27, wherein the device for contacting one or more samples comprises a fluidic system.

[0091] 29. A method for detecting one or more biomolecular targets, comprising: a. i. A plasmonic nanostructure biosensor comprising a substrate and a metal film disposed on the substrate as defined in any one of items 1 to 12 or as defined in item 25, wherein the metal film comprises a plurality of nanoelements arranged in a predetermined pattern, each of the nanoelements having a dimension of less than 100 nm, preferably 1 nm to 15 nm, more preferably 5 nm to 10 nm; ii. an apparatus for contacting one or more samples containing one or more biomolecular targets with the metal film surface(s) of the plasmonic nanostructure biosensor; iii. an incident light source for irradiating the surface of the metal film to generate the surface plasmons; iv. an optical detection system for collecting and measuring light displaced from the illuminated metal film, the displaced light being indicative of surface plasmon resonance on one or more surfaces of the metal film; and providing a plasmonic nanostructure biosensor system comprising: b. contacting one or more samples containing one or more biomolecular targets with the metal film surface of the plasmonic nanostructure biosensor; c. illuminating one or more surfaces of the metal film of the plasmonic nanostructure biosensor with an incident light source to generate surface plasmons before and after contact with one or more samples; d. collecting and measuring light displaced from the illuminated film with an optical detection system before and after contact with one or more samples; e. detecting one or more biomolecular targets based on a change or difference in the measurement of light displaced from the illuminated membrane before and after contact with one or more samples; A method comprising:

[0092] 30. The method of item 29, wherein the biomolecular target is a eukaryotic cell, a eukaryotic cell component, a prokaryotic cell, a prokaryotic cell component, a virus particle, a protein, an oligonucleotide, a prion, a toxin, or any combination thereof.

[0093] 31. The method of any one of items 29 or 30, wherein the collected light comprises light in a transmission mode, a reflection mode, or a combination thereof.

[0094] 32. The method of any one of items 29 or 31, wherein the step of measuring the displaced light includes measuring the light over a spectral range selected to include at least one plasmon band.

[0095] 33. The method of any one of items 29 or 32, wherein the change in the measurement of displaced light before and after contact is a resonance peak shift, a change in resonance peak intensity, a broadening of the resonance peak, a distortion of the peak resonance, or a change in refractive index.

Claims

1. 1. A method of manufacturing a plasmonic nanostructure biosensor, the plasmonic nanostructure comprising a substrate and a metal film disposed on the substrate, the method comprising: i. a first layer of one of said metal films is deposited on said substrate under normal incidence of an evaporation flux to a thickness below optimal plasmon coupling; ii. The second layer is deposited using oblique deposition at a fixed deposition angle; disposing the metal film on the substrate under two deposition conditions: the second layer is deposited using glancing angle deposition at a fixed deposition angle of 80° to 90°; the first layer has a thickness of 40 nm to 65 nm, and the second layer has a thickness in the range of 1 nm to 30 nm; The method, wherein the total thickness of the first layer and the second layer is in the range of 65 nm to 80 nm.

2. The substrate is made of silicon, silicon dioxide, silicon nitride, glass, diamond, quartz, magnesium fluoride (MgF ２ ), calcium fluoride (CaF ２ ), ZnSe, germanium, or a polymer.

3. 3. The method of claim 1 or 2, wherein the metal film comprises or is made from a noble metal, a transition metal, or an alkali metal, or any combination thereof.

4. The method of any one of claims 1 to 3, wherein the substrate is a one-dimensional or two-dimensional nanocrystal template (ie, one-dimensional nanocrystals of a Blu-ray disc).

5. The method of any one of claims 1 to 4, wherein the metal is gold.

6. 6. The method of any one of claims 1 to 5, wherein the first layer has a thickness of 50 nm to 65 nm, more preferably about 60 nm, and the second layer has a thickness in the range of 1 nm to 20 nm, more preferably 1 nm to 15 nm.

7. The method of claim 6 , wherein the first layer and the second layer have a total thickness of about 70 nm.

8. 8. The method of any one of claims 1 to 7, wherein the metal disposed on the substrate is gold and the second layer is deposited using glancing angle evaporation, the first layer having a thickness of 40 nm to 65 nm, preferably 50 nm to 65 nm, more preferably about 60 nm, the second layer having a thickness in the range of 1 nm to 30 nm, preferably 1 nm to 20 nm, more preferably 1 nm to 15 nm, and the total thickness of the first and second layers being in the range of 65 nm to 80 nm, preferably about 70 nm.

9. A plasmonic nanostructure biosensor obtainable according to the method of any one of claims 1 to 8.

10. 10. In vitro use of the plasmonic nanostructure of claim 9 for detecting one or more biomolecular targets, preferably wherein the biorecognition event is less than 100 nm, preferably between 10 nm and 15 nm.

11. 1. A plasmonic nanostructure biosensor system for the detection of one or more biomolecular targets, preferably comprising: a. A plasmonic nanostructure biosensor comprising a substrate according to claim 9 and a metal film disposed on the substrate, wherein the metal film comprises a plurality of nanoelements arranged in a predetermined pattern, each of the nanoelements having a dimension of less than 100 nm, preferably 1 nm to 15 nm, more preferably 5 nm to 10 nm; b. A device for contacting one or more samples containing one or more biomolecular targets with the metal film surface(s) of said plasmonic nanostructure biosensor; c) an incident light source for irradiating the surface of the metal film to generate surface plasmons; d. an optical detection system for collecting and measuring light displaced from the illuminated metal film, the displaced light being indicative of surface plasmon resonance on one or more surfaces of the metal film; A plasmonic nanostructure biosensor system comprising:

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