巴西专利BR112014000850B1 sensor device and sensor system

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专利摘要:
SENSOR DEVICE AND SENSOR SYSTEM The invention relates to a sensor device comprising a dielectric substrate (52) and a metallic layer (53) on the substrate (52) with at least one series of cavities (54) in it and adapted to support L- SPR, each cavity (54) in the metallic layer (53) has an opening (56) and a closed base (58) and an extension from the opening to the base. A layer of dielectric material (62) is fitted over the base (58) of each cavity (54) to reduce the apparent depth, the surface of the layer (62) is functionalized to connect the receiver sections (64). The sensor device is particularly designed for SPR detection but can be used in other detection techniques.
公开号:BR112014000850B1
申请号:R112014000850-7
申请日:2012-05-31
公开日:2020-11-10
发明作者:Andrea Valsesia；Franco Marabelli；Silvia Giudicatti；Gerardo Marchesini；François Rossi；Pascal Colpo
申请人:Plasmore Srl；The European Union, Representada Pela European Commission；
IPC主号:

专利说明:

Field of the Invention
[001] The present invention relates, in general, to sensor systems based on plasmon resonance on the surface, and more specifically, to the SPR sensor device with a nanostructure suitable for use, that is, in chemical, biochemical, biological tests. , biomedical, pharmaceutical and physical. Fundamentals of the Invention
[002] There are several known sensors that use the activation of plasmons on the surface, called Plasmonic Resonance Surface Sensors (SPR), to detect changes in the refractive index in a sample adjacent to the sensor surface. SPR sensors are used, for example, to quantify concentrations of substances in chemical, biochemical, biological, biomedical or pharmaceutical research in clinical or food diagnosis or in measurements in the environment (ie, detection of gas or waste water), etc. Several SPR sensors can perform large inspections quickly and in parallel, which makes them also useful when quantifying molecular interactions, particularly in the study of affinity and reaction kinetics in real time between two or more interactable molecules.
[003] SPR sensors are based on well-known SPR phenomena that involve one or more electromagnetic waves fixed on the surface and propagated at the interface between the metallic material (usually gold or silver) and the dielectric material. Each electromagnetic wave fixed on the surface, which occurs through the collective oscillation of free electrons at the metallic-dielectric interface, propagates at its highest intensity and parallel to the aforementioned interface and fades exponentially at the interface.
[004] The most commonly used techniques that trigger the SPR exploit a prism in the Kretschmann configuration. In this case, the prism is covered by plasmons on the surface that support a noble metal layer, and the SPR is optically driven by the prism. In fact, light can trigger the resonance of plasmons on the surface at the metallic-dielectric interface when a parallel component at the incident light interface and the electromagnetic wave fixed on the SPR surface have compatible frequencies and wavelengths. In the condition of resonance, the incident light is absorbed by the metallic-dielectric interface, to couple with the electromagnetic wave fixed on the surface. In this way, it is possible to observe the absorption when detecting, for example, a reduction in the intensity of the light that is transmitted or reflected by the metallic-dielectric interface. The coupling condition between light and plasmonic waves on the surface is very sensitive to changes in the refractive index of the dielectric medium close to the metallic-dielectric interface, and SPR sensors take advantage of the sensitivity in the coupling condition of the resonance to detect changes in the refractive index of the dielectric medium when measuring the decrease in the intensity of light reflected by the metallic-dielectric interface, at the same time that the latter is illuminated by the light beam activated by the SPR.
[005] SPR is used, in particular, in biosensor systems capable of detecting interactions between biomolecules or biochemical molecules, such as those between antigens and antibodies, enzymes and soil substances, endocrines and receptors, nucleic acids, etc. . In particular, several SPR biosensor systems have bioreceptors attached to the surface of their sensor, to detect changes in the condition of light coupling triggered by the SPR and arising from changes in the refractive index on the sensor surface when the biochemical or biomolecules interact (or unite) with the bioreceptors. The biosensor systems are suitable for measuring, for example, the concentrations of biomolecules or biochemical molecules in solutions, etc.
[006] Currently, there is a multitude of laboratory equipment based on SPR sensors. US patent application No. 2009 / 021,727 describes biosensors based on the Kretschmann configuration.
[007] US 2008 / 316.490 describes another SPR biosensor system to detect biochemical molecules and which uses a metallic grid instead of a prism.
[008] More recently, the discovery of phenomena in localized surface plasmon resonance (L-SPR) and the expansion of transmission by the metallic and periodic structures of the wave sub-length is promising when it significantly increases the size of the detection series, presenting high yield uses. When using L-SPR, the simplest and most versatile technology that has been explored in a wide range of technological areas is the sensor configuration of the nano-opening series. In the classic approach, the SPR sensor consists of a dielectric substrate covered by a layer of noble metal in which a periodic series of nano-openings is formed, that is, openings that have dimensions in the wave sub-length.
[009] Sensors based on L-SPR with series of nano-openings are, for example, described in W02008 / 039212, W02010 / 130045 and by Parsons, J. et al. in “Localized surface-plasmon resonances in periodic non-diffracting metallic nanoparticle and nanohole arrays” (PHYSICAL REVIEW B 79, 073412 (2009)).
[010] Giudicatti, S. et al. in “Plasmonic resonances in nanostructured gold / polymer surfaces by colloidal lithography”, PHYSICA STATUS SOLIDI (A), vol. 297, April 2010 (2010-04), pages 935-942 describes a colloidal lithography procedure for preparing an LSPR support structure consisting of a golden film perforated by polymeric columns and arranged in a 2D hexagonal series.
[θll] The use of colloidal lithography in the preparation of SPR sensors is also presented in EP 2 264 438; and in “Bioadhesive nanoareas in antifouling matrix for highly efficient affinity sensors” by Mannelli et al., PROCEEDINGS OF THE SPIE - USA, vol. 7035, 2008, pages 70350Y- 1-70350Y-10. Object of the Invention
[012] The purpose of the present invention is to propose a series nano-opening sensor device for SPR sensor with increased sensitivity.
[013] The said objective is achieved by a sensor device in the manner described here. Summary of the Invention
[014] During the work related to the above invention, the inventors observed that although current technologies allow to adapt the geometry of nano-openings in a way compatible with the electromagnetic response, the resulting geometry may not be optimal in relation to the biological detection process. In particular, the inventors have observed that the position of biomolecular receptors fixed on the metal and the openings are often poorly located to ensure the most effective detection.
[015] Considering the problem above, the inventors created a plasmonic sensor device with a structure that ensures that the binding of a ligand / analyte in a receptor occurs in a region with a strong density in the electric field.
[016] For the above reasons, the sensor device, in particular the SPR, according to the present invention, comprises a dielectric substrate and a metallic layer on the substrate adapted to support the L-SPR. The metallic layer has at least a series of cavities in it, each has a depth, an opening and a closed base, and the cavities expand from the opening to the base.
[017] It should be noted that the layer of dielectric material is supplied on the base of each cavity, so that it offers a reduced depth (the apparent depth) from the opening, the surface of the layer is functionalized so that there is bonding affinity with the receiver portions.
[018] The first aspect to be observed concerns the way of enlarging the cavity that strengthens the electric field around its opening. Then, so that the receivers are closer to the region above, and the connection occurs in the region of high density, the base of the cavity is filled by the dielectric material that forms a layer, reducing the apparent depth of the cavity. Finally, the apparent depth corresponds to the residual depth between the top / opening of the cavity and the surface of the layer.
[019] In addition, the functionalized surface of the layer allows it to fix the portions of the receptor, which, during the tests and tests, are connected with the corresponding analytes / ligands that may be present in the tested sample.
[020] With the procedure, the portion of the receiver approaches the strong electric field and the analyte connected to it comes to exist in the region of the strong electric field.
[021] The procedure designed above makes the cavities, in the form presented, compatible with the target electromagnetic specifications, while optimizing the position of the receiver portions and the complex that is formed in use for optimal detection.
[022] In the form used here, the expression “receptor portion” indicates any type of substance to be attached to the sensor, such as a probe substance, and which has a specific binding specificity in relation to the analyte to be detected. The receptor portion can be any molecule, chemical, natural or biological substance or part; in biotests, portions of the receptor may comprise the following: antigens / antibodies, enzymes, proteins, oligonucleotides, etc. The term “analyte”, on the other hand, indicates any molecule, chemical substance, biomolecule or compound of interest to be detected by the present sensor as a result of the specificity of binding of the analyte with the portion of the receptor fixed in the sensor device. The analyte can be any type of ligand, molecule, biological substance, that is, proteins, enzymes, peptides, organic and inorganic chemicals, oligonucleotides, antibodies, etc., in general any type of molecule that can be recognized by a biochemical reaction specific.
[023] Regarding the shape of the nanocavities, continuous, frustoconic or graduated trapezoidal shapes are preferred. However, those skilled in the art can create any suitable shape, enlarged from the top to the bottom, resulting in the pattern of a strong electric field around the opening of the cavity.
[024] As noted below, the apparent depth of the cavities is adapted according to the type and, in particular, depending on the length, portion of the receiver and analyte. The thickness of the electrical layer in the cavity is advantageously designed so that when the analyte is connected to the receiver portion, it is at least partially located in the region of the strongest electric field. This implies that the connecting end of the receiver portion is contiguous to the region of the strongest electric field or partially penetrates it. Following the reasoning, it can be observed that the layer generally has a thickness (or height, that is, the distance from the base of the cavity to the surface of the top of the layer) that is less than the depth of the cavity (or height).
[025] In biological uses, the thickness of the layer is preferably such that the apparent depth is in the range of 10 to 30 nm from the opening, more preferably between 15 and 25 nm.
[026] The layer can be made of any suitable dielectric material that can be functionalized with or at the receptor portion adapted to detect the analyte in question. The layer can, for example, be made of ppAA or nylon.
[027] It should also be noted that the layer can be made of a single material or comprise two or more layers of dielectric material, where the thickness of each layer contributes to the total thickness of the layer, allowing to adjust the apparent depth of the cavity . In this case, the topmost layer must have the capacity for functionalization. For example, the first layer of dielectric material, such as ppAA or nylon, can be formed with a thickness of tl; and a subsequent t2-thick layer of any functionalized dielectric material, i.e., porous dielectric material, including hydrogels, can be formed on the first layer. The total thickness T of the layer is T = tl + t2; and where the depth of the cavity in the metallic layer is dc, the apparent depth dA is calculated as dA = dc - T.
[028] Following the above reasoning, it can also be observed that the functionalized layer can include molecules / portions that form the portions of the receptor, it is not necessary to locate the portions of the receptor on the substrate before use.
[029] In one embodiment, the metallic layer has a thickness of at least 100 nm, preferably at least 120 nm. In practice, the metallic layer can have a thickness in the range of 100 to 200 nm.
[030] The dimensions of the cavities are in the nanometric range and, as is known, to stimulate the plasmons on the surface, the cavities have dimensions in the wave sub-length, that is, below that of the incident light.
[031] The cavities in the metallic layer are usually perforated, from where its closed base is formed by the substrate surface. The cavities in said layer preferably have a depth of at least 100 nm and, more preferably, in the range of 100 to 200 nm.
[032] In addition, in general operations with selected probe light from the UV-VIS-NIR spectrum, the cavities can be designed to have an opening width (ie diameter or equivalent diameter) in the range of 50-250 nm, whereas the base width can be in the range of 100-450 nm.
[033] In this context too, the periodicity of the nanocavity series, that is, the distance between the center of two cavities, can be in the range of 200 to 1,000 nm.
[034] Any metal adapted for L-SPR can be used in the metallic layer that supports the L-SPR. The metal can be selected from the group comprising the following: gold, silver, copper, platinum, aluminum or alloy comprising one or more of these minerals.
[035] Advantageously, the metallic layer is covered by a thin anti-fouling layer that prevents the adhesion of proteins. As a result, fixing the receiver portions on the surface of the dielectric layer, with the functionalized surface, is easier. And during the tests, there is less adhesion of proteins and analytes to the metal, which improves the signal-to-noise ratio.
[036] In the context of the present invention, the term “dielectric (a)”, in the form used in relation to the substrate and the layer material, includes materials that are not “conductors” and that can be polarized by the plasmonic field and, for this reason, they comprise dielectric materials, such as sapphire, glass or polymers, in addition to suitable semiconductor materials.
[037] To facilitate use, the microfluidic layer can be supplied on top of the metallic layer, common in the state of the art. The microfluidic layer generally comprises channels arranged to selectively put samples in contact with a respective series of cavities.
[038] Generally, a series of cavities is associated with a type of receptor.
[039] The metallic layer comprises a plurality of series of cavities in which each series comprises a respective type of portion of the receiver.
[040] This sensor can be used in the SPR analysis system with conventional lighting and light analysis system, either with the configuration of photodetectors or the imaging system.
[041] Following the above reasoning, it can be seen that when using SPR images / microscopy, SPR presents a substantially higher performance in relation to conventional SPR and L-SPR sensors. It is known that with conventional sensors, the probe light is selected as a trade-off between sensitivity and lateral resolution. In contrast, it is observed that in the SPR sensor, which uses series of nanocavities, plasmons are confined in a strict area, leading to fine lateral resolution (usually between 100 and 500 nm). In addition, the cavity structure with the layer that reduces the depth of the cavity allows the target portions to be positioned in the strong electric field for maximum sensitivity. As a result, SPR images can be executed in high lateral resolution and with high sensitivity.
[042] The sensor device can be used in a large number of SPR analyzes, depending on the type of receiver portions fixed on the layer surface. Among other uses, the SPR sensor device can be used for tests in the chemical, biochemical, biological, biomedical, pharmaceutical and physical fields. More particularly, SPR sensors can be used, for example, to quantify concentrations of substances in chemical, biochemical, biological, biomedical or pharmaceutical research, clinical or food diagnostics or in measurements in the environment (ie detection of gas or waste water) ), etc.
[043] It is also noted that the structure of the sensor device can be interesting in molecular, chemical and / or biochemical detection techniques in addition to detection systems based on plasmonic resonance of the surface. In particular, the sensor can be used advantageously in detection / analysis techniques in which the location and magnification of the magnetic field, indicated in the present invention, are reflected in the amplification of the detection signal, which can be optical, thermal, electrical, etc. Examples of techniques are as follows: a. Surface Amplified Raman Spectroscopy (SERS). In standard Raman spectroscopy, copying the monochrome laser light triggers the vibrational modes of the molecules. The modes can be seen in the spectrum of reflected light as positive peaks. The spectral position of the peaks (the Raman Spectrum) is characteristic of the molecule (like a fingerprint). Raman Spectroscopy is the only way to directly determine the presence and quantity of the different molecules present in a compound. In conventional SERS, the surface of the nanostructured sensor amplifies the Raman signal (which is usually very weak), making it possible to determine substances in lower concentrations. The sensor device according to the invention can be used advantageously at SERS. In this case, the SERS enlargement (as in the Surface Plasmonic Resonance technique) occurs at the top of the cavity and can be located when the size and shape of the cavities are improved. Just as in the case of the L-SPR analysis, with the aforementioned sensor device it is possible to locate the molecules to be detected exactly when the SERS magnification reaches its maximum, increasing the signal sensitivity. B. Fluorescent Spectroscopy. In standard fluorescent spectroscopy, the fluorescent molecule (fluorophore) is triggered by the laser light and emits fluorescent light at a different wavelength. The technique is used as a method of quantitative analysis to determine the concentration of molecules, proteins, DNA, etc. With the present invention, the fluorophore (combined with the secondary antibody or the DNA sequence that makes the recognition) is located in the region where the electric field reaches its maximum, and the sensitivity (that is, the intensity of the fluorescent light) can be expanded significantly. ç. In Matrix Assisted Laser Ionization and Desorption (MALDI) and Surface Assisted Laser Ionization and Desorption (SALDI), a high density leisure beam is used to induce desorption of surface analytes to create ions that can be analyzed by a Mass Spectrometer. The analysis technique is very sensitive and specific to directly determine the concentration of the molecule. When using the sensor device in SALDI, the molecules to be analyzed are located where the intensity of the laser light (which induces desorption) reaches its maximum, making the analysis more effective.
[044] It can be seen that in the different analysis techniques, the plasmons on the surface are also stimulated locally by the incident light and an enlarged electric field is obtained at the opening of the cavities thanks to their specific shape, as "only" in the SPR analysis. Brief Description of Drawings
[045] The present invention is described below by way of example and with reference to the following attached drawings: - FIG. 1 is a basic drawing of the conventional SPR sensor with a series of nano-openings; FIG. 2 is a drawing illustrating the design principle of the SPR sensor device; FIG. 3 is a comparative drawing (not a SPR sensor modality) that illustrates the location of the receptor portion at the base of the cavity without the dielectric layer; FIG. 4 is a view of the nanostructure layout according to a SPR sensor modality; FIG. 5 is a view illustrating the intensity pattern of the electric field within and around the nanostructure; FIG. 6 is a basic diagram of a SPR sensor system modality. Detailed Description of a Preferred Mode
[046] a) Conventional model of the L-SPR sensor
[047] As is known to those skilled in the art, sensors based on the SPR effect are based on two types of plasmon resonance effect:
[048] 1) Sensors based on Plasmon-Surface Polars (SPP) that are undulations at the interface between a dielectric and noble metal;
[049] 2) Sensors based on Plasonic Resonances of Localized Surface (L-SPR) that are standing waves located inside dielectric nanocavities in a noble metal material or the equivalent of standing waves located inside noble metal nanostructures inside or in surface of a dielectric medium.
[050] In the two cases mentioned above, of course, the sensors can detect variations in the refractive index at the metallic interface.
[051] It should be noted that in SPR biosensors the microscopic (or local) sensitivity determines the general sensitivity of the sensor. The local sensitivity is related to the length of the electric field extension of the plasmon resonance in the dielectric region that surrounds the metal. The length is called the depth of penetration.
[052] Generally, the penetration depth of the L-SPR has a magnitude of the order of 10 nm. Numerical simulation makes it possible to evaluate and study the pattern of distribution of the intensity of the electric field around plasma nanostructures.
[053] In the conventional design of sensors based on L-SPR with series of nano-openings, as, for example, shown in Fig. 1, cylindrical openings / cavities 2 (that is, by ion beam) are drilled into the metallic layer that supports plasmon resonance 4 disposed on the dielectric substrate 6. In this case, the electric field pattern in cavity 2 is maximum along the cylindrical walls, as represented by the dark gray vertical rectangles 8 in Fig. 1. The normal distance from the wall of the strongest electric field is around 10 nm.
[054] In the uses of the biosensor, the portions of the receptor, that is, proteins, are generally immobilized in the cavities, the portions show a specificity of binding in the analytes to be detected in the sample. Generally, the receiver's portions are fixed on the cavity walls where the electric field is maximum.
[055] However, the receiver portions have finite dimensions; antibodies, for example, have a length that generally ranges from 10 to 20 nm.
[056] In addition to the above, it is observed that, with an electric field around 10 nm, the receiver portion is effectively located in the region where the plasmon resonance electric field is maximum.
[057] When the analyte reaches the cavity, it is recognized by the biosonde and immobilized on its receptor creating a complex.
[058] The analyte, however, is located in the region of cavity 2, which is relatively outside the maximum intensity of the electric field, so as not to be detected with the optimal sensitivity.
[059] This situation is illustrated in Fig. 1 in which the indicated antibodies 10 that form the receptor portions are fixed on the side walls of the cavity 2. The extent of the antibodies 10 can be compared with the width of the region of the strong electric field 8. Reference signal 12 indicates an antigen bound on the antibody of a receptor 10. Analyte 12 present in the test sample has a unique binding specificity on antibody 10 and combined with it forms a complex. However, due to the reduced extent of the strong electric field, the connection takes place outside it.
[060] b) Operating principle of the sensor
[061] The concept of the projected sensor is illustrated in Fig. 2. To optimize the sensitivity of the L-SPR sensor, the inventors configured the geometry of the cavities to obtain a strong and expanded electric field pattern in a region of the cavity, in addition to identify a means to arrange the portions of the receiver contiguously to the region of strong electric field, or with partial overlap in it, so that the analytes linked in the portions of the receiver are located in the strong electric field and, consequently, in a region of maximum sensitivity / expanded.
[062] With reference to Fig. 2, the SPR 50 sensor device comprises a dielectric substrate 52 with an L-SPR support layer comprising a noble metal layer 53 on the dielectric substrate 52 with a series of nanocavities 54 in it (only some wells 54 are shown here for simplification purposes). The dimensions of the cavities 54 are generally referred to as "sub-wavelength", that is, they are less than the wavelength of the probe's light beam. The cavities 54 are created asymmetrically, that is, their cross-section is not constant across the depth of the cavity so that it is asymmetrical in the direction of the incoming light beam. However, more specifically, the cavities 14 have a shape that extends from the opening 56 to the base 58, the closed base of the cavity is formed by the surface of the underlying substrate 52. The enlarged shape can be continuous, as in Fig. 2, that is, based on the shape of a truncated pyramid or cone, or projected as a graduated profile. The depth of the cavity 54, indicated as dC, represents the vertical distance from the opening 56 to the base 58. When the SPR sensor is illuminated with the probe light from the side of the substrate, that is, from the wider side of the base 58, at an appropriate angle and wavelength, plasmon resonance is triggered. The field strength of the plasma-polyaritons on the surface is potent and reaches maximum levels in the opening section 56 of the cavity, as illustrated by rectangle 60 in Fig. 2.
[063] The ability to adapt the magnetic response in nano-open series is well known in the art (see, as an example, the article by Li, J. et al. “Studies of the plasmonic properties of two-dimensional metallic nanobottle arrays ”In APPLIED PHYSICS LETTERS 92, 213106 (2008)). In fact, the triggering and radiation of plasmon-polyaritons on the surface depends greatly on geometry; resonance depends on the size and shape of the metallic nanostructure. It has been shown that the spectral position of plasmon-polyaritons on the surface depends mainly on the period of the series of nanocavities, while the strength and field pattern depend largely on the effective shape of the cavity arranged in the metallic layer.
[064] As mentioned above, the asymmetric shape of the cavity 54 (enlarged from the opening to the base) leads to the concentration of the strongest electric field around the opening of the cavity 56. The geometry / dimensions of the cavity 54 are chosen so that obtain the targeted electromagnetic response.
[065] It should be noted that the sensor device 50 also includes a layer 62 of dielectric material disposed at the base 58 of the cavity 54 which reduces the apparent depth of the cavity 54. Consequently, while the opening in the metallic layer 53 retains a dC depth ( corresponding to the thickness of the metallic layer - the cavity is an orifice), the base of the cavity is filled with dielectric material and appears to have a dA depth, representing the distance from the top / opening 56 to the surface of layer 62. Layer 62 forms a type of support structure or pedestal on whose surface 63 the portions of the receiver 64 can be fixed in the vicinity of the region of the strong electric field. Consequently, when the corresponding analyte binds to the receptor portion, it is located in the region of maximum sensitivity. In other words, the cavity 54 is partially filled with the dielectric material that makes up layer 62, so that it opens from the top of the metallic layer and is hollow at the depth corresponding to the apparent depth dA.
[066] The above situation is illustrated in Fig. 2 in which the antibody of receptor 64 is fixed on the surface 63 of layer 62 and presents its opposite end in interface with the pattern region of the strong electric field 60. In connection with the antibody 64, the coagent antigen 66 extends in the region of the maximum electric field 60 and in the region of maximum sensitivity.
[067] For comparison purposes, Fig. 3 illustrates the structure of the SPR sensor designed with a conical 54 'cavity and similar to that of the sensor in Fig. 2, without, however, the dielectric layer. As can be seen, the 64 'antibody is located at the base 58' of the cavity itself, and even at the connection, the complex formed is well away from the region of the strong 60 'electric field. It should be noted that in said nanostructures the depth of the opening dC is generally greater than 100 nm, while the normal size of an antibody is 15 nm.
[068] Returning to the sensor, as shown in Fig. 2, the fixation of the receiver portions is usually obtained by the functionalized surface of the material that forms layer 62. The functionalization of the material is performed to present a selective bonding of the targeted type of the portions of the receiver, the latter are selected depending on the type of test to be performed. The material suitable for the functionalized polymer is the polyacrylic-acid or dielectric polymer with functionalities suitable for optimized fixation of the target portions.
[069] Although in Figures 2 and 3 layer 62 is shown as a single layer, it can comprise two or more layers of dielectric material. For example, the first layer of dielectric material can be arranged at the base of the cavity and its second layer is formed on top of the first. In this case, the upper surface of the second (or higher) layer forms the surface of the layer, and its thickness is the total of the individual thicknesses of each layer. The dielectric material of the second layer (or higher) has the intended functionalization to fix the probes (portions of the receiver); porous dielectric materials, including hydrogels, can be used in the second / top layer.
[070] The practical mode of the sensor device is illustrated in Fig. 4. Similar characteristics are indicated with the same reference signals, as in Fig. 2, increased by 100. The sensor device 150 is set according to the design principle above. The sensor device 150 comprises a dielectric substrate 152 made of glass. The noble metal layer, in this case, gold, is indicated 153 and arranged on the glass substrate 152. The layer comprises a series of cavities 154 formed in the golden layer, although only one is shown for simplification purposes. The cavity 154 in the metal has a depth indicated as dC. It has an enlarged and graduated cross section that consists of three sections: an opening section with radius R1, an intermediate section with maximum radius R2 and a final section with maximum radius R3. It can be seen that the cavity opening 156 is configured as a collar section 170 slightly protruding from the surface of the metal layer 153.
[071] Cavity 154 is partially filled with dielectric material forming a layer 162 that reduces the apparent depth of the cavity at depth dA. The surface of layer 162 is functionalized so that the portions of the receptor are fixed on it, as illustrated by the antibodies 164 represented in Fig. 4.
[072] For purposes of example, it is appropriate to comment on materials and dimensions, but they should not be interpreted in a limited way. Instead of the glass substrate 152, other transparent dielectric materials can be used, that is, transparent polystyrene or PMMA or polycarbonate; alternatively, a semiconductor material can be used, preferably with low doping. Furthermore, instead of gold, other metals, i.e. noble metals, such as Pt or Al, can be used. The dielectric material of layer 162 can be ppAA or any other suitable and functionalized polymer. The golden layer has a thickness of the order of 120 nm around the opening of the cavity; the depth of the dC cavity is 120 nm. The thickness of layer 162 is 100 nm where the apparent depth dA is 20 nm. The opening radius is RI = 50 nm, while the base of the cavity is R3 = 200 nm. To recapitulate, the dimensions are presented for the purpose of exemplification and should at no time be interpreted in a limited way.
[073] The electric field pattern in the plasmon resonance of the nanostructure in Fig. 4 is determined by the numerical simulation and the distribution of the electric field is represented in Fig. 5. As can be seen, considering the above indications about shapes and dimensions, there is a region of strong electric field around the opening section 156 of the cavity; due to the height of the layer and the position of the receptor portions, the analyte attached to the receptor portion is located in the region of maximum sensitivity.
[074] Preferably, a thin layer of anti-fouling material 172 is formed on the metal surface (and not on the surface of layer 163). As is known, the anti-fouling material acts in an anti-adhesive manner, so that unwanted interactions are reduced or eliminated, such as the non-specific absorption of chemical or biochemical molecules, etc. This reduces the noise signals that may come from the interaction or binding of unwanted chemical or biochemical molecules on the sensor surface.
[075] c) Sensor manufacture
[076] The sensor can be easily manufactured using lithographic techniques. A possible manufacturing technique is described below. A layer of polyacrylic acid (ppAA) is deposited on the glass substrate; and a subsequent layer of polystyrene (PS) beads is deposited on the ppAA. The layers of ppAA and PS are etched by plasma 02, to form a grid structure that comprises pillars of ppAA spaced regularly and separated by a submicrometric distance. The pillars have a conical shape from the base to the top. The gold is deposited on the pillars to fill the spaces between the surrounding pillars, and the rest of the PS mask is removed, obtaining a periodic golden nanogrid. Other possible materials for dielectric abutments are polystyrene or polymethylmethacrylate, etc.
[077] It can be seen that, at that stage, the cavities are completely filled with the dielectric material of the columns. To form the cavities with the layer that fixes and locates the portions of the receiver, it is sufficient to selectively engrave (in gold) the top of the columns to the apparent depth dA. This technique has the advantage that the ppAA recording is performed with great precision, that is, around +2 nm.
[078] The thin layer of anti-fouling material (ie anti-fouling hydrogel, ie polyethylene glycol) can be selectively deposited on the golden surfaces and exposed.
[079] d) Use in the SPR sensor system
[080] With reference to Fig. 6, it schematically represents the SPR sensor system which comprises the SPR sensor device in the manner described above. In operation, the sensor is directed so that the light from the probe falls on the side of the substrate 252 and illuminates the cavities 254 on the broader bottom side, that is, opposite the opening. Generally, the sensor device comprises at least a series of 254 cavities designed according to the principle that underpins the L-SPR, reaching the aforementioned electric field pattern.
[081] In the practical modality, the metallic layer 253 is covered by a microfluidic system 280 comprising channels that put the tested sample in contact with a series of respective cavities 254. In this case, each series can be prepared to contain different portions of the receptor and detect different substances. The microfluidic system 280 can be made by forming a layer of elastomer or other materials (i.e., PDMS, PMMA, glass, epoxy, etc.) on the top of the metallic layer 253 with channels 282 formed therein, as known in the art.
[082] When used, the SPR sensor device is illuminated from the side of the substrate by a beam of light 290 (beam of probe light) coming from the optical input module 292. The beam of light 290 has a recognized frequency and incident angle by triggering the SPR on the sensor surface. It can be seen that the beam of light 290 that illuminates the series of nanocavities can be configured in the way already known, to present a certain polarization. To activate the SPR, the light beam 290 has one or more frequencies that match one or more permitted frequencies of the electromagnetic waves fixed on the surface in the sensor's nanostructure. Preferably, the sensor is designed so that the permitted frequencies of the SPR generally correspond to the visible / near-infrared light spectrum. While the light beam activates the SPR, at least part of the light beam is absorbed on the surface of the sensor where the extent of absorption depends on the frequency of the light incident on the surface of the sensor. The light from the light beam that is not absorbed on the surface of the sensor is reflected and intercepted in the optical output module 294. It can be seen that operation in transmission mode is also possible.
[083] The light property of the reflected light beam, preferably its intensity, is detected (measured) in the optical output module 294 and the effective value of the measured light property which represents the level of activation of the plasmons on the surface and that allows to assess the state of resonance or the change of its established condition in relation to the calibrated or previously stored / acquired data. As is known, the resonance condition generally leads to a decrease in the measured intensity of the light reflected from the light beam due to the absorption of light on the sensor surface, and modification of the refractive index of the sample adjacent to the sensor surface alters the condition of resonance.
[084] As is known to the person skilled in the art, the detection / measurement of the optical output module can be performed by a photodetector or a series of images.
[085] d) Use of the SPR sensor in other detection techniques
[086] As explained above, the sensor device can be used advantageously in other detection techniques. In fact, the L-SPR effect (oscillation of electrons from the noble metal layer located just around the nanocavity) produces, combined with the conical shape of the nanocavities, an enlarged electric field around the cavity opening, which can be beneficial in other analysis techniques, such as Raman / SERS, fluorescent spectroscopy and SALDI.
[087] In fact, the electric field plays a role in all the aforementioned uses and the following can be observed: - in Raman spectroscopy (SERS), the electric field determines the strength of the Raman emission and, consequently, its signal and sensitivity. - in fluorescent spectroscopy, the electric field (at a special wavelength) is what drives the fluorescent molecules to emit light. As a consequence, the location of the fluorescent molecule at the top of the cavity, where the electric field reaches its maximum, acts by amplifying the emitted light, as well as the sensitivity. - in SALDI, the proteins to be analyzed by Mass Spectrometry are induced to be absorbed with the use of a high-energy laser that increases the temperature locally. The energy of the laser beam (the thermal energy it can release on the surface) is proportional to the strength of the electric field. By using the sensor described above, it is possible to concentrate the electric field in which the molecules are located and to optimize desorption.

权利要求:
Claims (20)
[0001]
1. Sensor device comprising the following: a dielectric substrate (52; 152; 252); a metallic layer (53; 153; 253) on the substrate (52; 152; 252) with at least a series of nanocavities (54; 154; 254) in it and adapted to support L-SPR, each cavity (54; 154; 254 ) in the metallic layer having a depth, an opening (56; 156) and a closed base (58; 158) and an extension from the opening to the base, to form a strong pattern of electric field around the opening of the cavity; characterized by a layer (62; 162) of dielectric material being provided on the base (58; 158) of each cavity (54; 154) to form a reduced and apparent depth (dA), the surface of the layer (63; 163 ) functionalized to connect the portions of the receiver (64; 164); the thickness of the dielectric layer being projected so that, with the connection of an analyte in the portions of the receiver, the analyte is partially located in the region of the strong electric field.
[0002]
Device according to claim 1, characterized in that the cavities (54) have a frustoconical or trapezoidal shape from the opening to the base.
[0003]
Device according to claim 1 or 2, characterized in that the expansion of the cavities (154) is continuous or in stages.
[0004]
Device according to any one of claims 1 to 3, characterized in that the cavities are closed at the base by the dielectric substrate.
[0005]
Device according to any one of claims 1 to 4, characterized in that the layer thickness is less than the depth of the cavity.
[0006]
Device according to any one of claims 1 to 5, characterized in that the height of the layer (62; 162) is such that the apparent depth (dA) is in the range of 10 to 30 nm from the opening (56) , preferably between 15 and 25 nm.
[0007]
Device according to any one of claims 1 to 6, characterized in that the layer (62; 162) consists of a functionalized dielectric polymer, at least on the surface.
[0008]
Device according to any one of claims 1 to 7, characterized in that the layer is made of a single dielectric polymer or of two or more layers of dielectric material, and the upper layer comprises a functionalized dielectric polymer.
[0009]
Device according to any one of claims 1 to 8, characterized in that the metallic layer (53; 153; 253) has a thickness of at least 100 nm, preferably at least 120 nm.
[0010]
Device according to any one of claims 1 to 9, characterized in that the metallic layer (53; 153) is made of a metal selected from the group comprising the following: gold, silver, copper, platinum, aluminum or a alloy comprising one or more of said materials.
[0011]
Device according to any one of claims 1 to 10, characterized in that the cavities have an opening amplitude in the range of 50 to 250 nm, a base amplitude in the range of 100 to 450 nm and a depth of cavity in the range of 100 to 200 nm.
[0012]
Device according to any one of claims 1 to 11, characterized in that at least one series of cavities has a periodicity in the range of 200 to 1,000 nm.
[0013]
Device according to any one of claims 1 to 12, characterized in that the metallic layer is covered by a layer of anti-fouling material (172).
[0014]
Device according to any one of claims 1 to 13, characterized in that it comprises portions of the receiver (64; 164) fixed on the surface of the layer.
[0015]
Device according to any one of claims 1 to 14, characterized in that it comprises a microfluidic layer (280) on top of the metallic layer (253), and the microfluidic layer comprises channels (282) arranged to selectively contact test samples with the respective series of cavities 254.
[0016]
Device according to any one of claims 1 to 15, characterized in that the substrate comprises dielectric material and / or semiconductor material.
[0017]
17. SPR sensor system characterized by comprising: a sensor device as defined in any one of claims 1 to 16; an optical input module (292) that emits at least one tested light beam (290) to illuminate the metal support layer L-SPR of the sensor device under conditions selected to activate the SPR; and an optical output module (294) for measuring the light property of at least one beam of light tested in the form transmitted or reflected by the sensor device, to monitor the condition of resonance on the sensor surface.
[0018]
System according to claim 17, characterized in that the sensor device is illuminated with at least one beam of light tested from the side of the dielectric substrate (252).
[0019]
19. Use of a sensor device as defined in any one of claims 1 to 16, characterized in that it is done in a detection method based on one or more techniques among SPR, SERS, fluorescence and SALDI.
[0020]
20. Sensor system comprising a device as defined in any one of claims 1 to 16, characterized in that it is configured to operate a chemical, molecular, biochemical or biomolecular detection based on SPR, SERS, fluorescence and / or SALDI.

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法律状态:
2018-12-11| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

2020-06-23| B09A| Decision: intention to grant|

2020-11-10| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 31/05/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

申请号 | 申请日 | 专利标题

EP11174058.5|2011-07-14|

EP11174058A|EP2546635A1|2011-07-14|2011-07-14|SPR sensor device with nanostructure|

PCT/EP2012/060310|WO2013007448A1|2011-07-14|2012-05-31|Spr sensor device with nanostructure|

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