Novel super-surface SPR chip and preparation method thereof

Abstract

The invention provides a novel super-surface SPR (Surface Plasmon Resonance) chip and a preparation method thereof, and belongs to the technical field of surface plasmon resonance sensors. The novel super-surface SPR chip comprises a substrate, a nanocup array and a metal film, the nanocup array is located on the substrate, and the metal film is evaporated on the surface of the nanocup array structure; the number of layers of the metal films is 2-11; when the number of layers of the metal film is two, the metal film is divided into a bottom layer and a covering layer; when the number of layers of the metal film is 3-11, the metal film is divided into a bottom layer, an interlayer and a covering layer; the metal of the bottom layer is selected from one of niobium pentoxide, tantalum pentoxide, zirconium oxide, hafnium oxide and titanium; the metal of the interlayer is selected from gold or silver; the metal of the covering layer is gold. The characteristics of different metals are fully utilized, the prepared novel super-surface SPR chip is higher in response signal and sensitivity, and biomarkers with lower concentration can be detected.

Description

Technical Field

[0001] The invention relates to the technical field of surface plasmon resonance sensors, and in particular to a novel supersurface SPR chip and a preparation method thereof. Background Art

[0002] In the field of molecular interactions, surface plasmon resonance (SPR) sensor technology has attracted widespread attention due to its high sensitivity and real-time monitoring capabilities. SPR sensors generate different resonance angles based on whether the ligand coupled to the metal surface binds to the analyte. By monitoring the changes in the resonance angle, the affinity, affinity constant, binding kinetics and other information of the biomolecular interaction can be inferred. It is widely used in the study of protein interactions, drug-receptor interactions, vaccine development, biosensing, biomembrane research, etc. At present, SPR sensors usually use gold (Au) or silver (Ag) thin films as the sensing layer. The thickness of the metal film has a significant effect on the sensor performance. The optimal thickness of the gold film is 40 nm. However, SPR sensors still face challenges in achieving higher sensitivity and stability. The following are the current problems or disadvantages of SPR sensors: (1) Sensitivity limitation: When the thickness of the gold film is between 45 and 50 nm, the SPR sensor has insufficient detection sensitivity for low-concentration biomarkers; (2) Material stability issues: Metal and metal oxide surfaces easily adsorb organic materials, which changes the interfacial free energy and affects the stability and sensitivity of the sensor; (3) Oxidation sensitivity: Although silver-based SPR sensors have superior performance, their sensitivity to oxidation and sulfurization limits their widespread application. Summary of the invention

[0003] In view of the above deficiencies in the prior art, the present invention provides a novel metasurface SPR chip and a preparation method thereof. By precisely adjusting and controlling the type, number of layers and thickness of metals in the SPR chip, a nanocup array metasurface SPR chip with multiple layers of metals is prepared, thereby solving the problems of insufficient sensitivity and stability of SPR sensors in the prior art, as well as oxidation sensitivity.

[0004] To achieve the above purpose, the specific technical solutions of the present invention are as follows:

[0005] A novel super-surface SPR chip comprises a substrate, a nanocup array and a metal film, wherein the nanocup array is located on the substrate, and the metal film is evaporated on the surface of the nanocup array structure;

[0006] The number of layers of the metal film is 2 to 11;

[0007] When the number of layers of the metal film is 2, the metal film is divided into a bottom layer (i.e., the first layer) and a cover layer (i.e., the last layer);

[0008] When the number of layers of the metal film is 3 to 11, the metal film is divided into a bottom layer (i.e., the first layer), an interlayer and a cover layer (i.e., the last layer);

[0009] The metal of the bottom layer is selected from one of niobium pentoxide, tantalum pentoxide, zirconium oxide, hafnium dioxide and titanium;

[0010] The metal of the interlayer is selected from gold or silver;

[0011] The metal of the covering layer is gold.

[0012] Preferably, the material of the substrate includes but is not limited to PET (polyethylene terephthalate) material, PP (polypropylene) material, PE (polyethylene) material, and PVC (polyvinyl chloride) material.

[0013] Preferably, the thickness of the substrate is 10-1000 μm.

[0014] Preferably, the period of the nanocup array is 300-500 nm, and the upper diameter of a single nanocup is 150-300 nm, the lower diameter is 50-200 nm, and the height is 200-600 nm.

[0015] Preferably, the total thickness of the metal film is 10-500 nm.

[0016] Preferably, when the number of layers of the metal film is 2, the metals of the bottom layer and the cover layer are titanium and gold respectively.

[0017] Preferably, when the number of layers of the metal film is 3, the metals of the bottom layer, interlayer and cover layer are respectively: titanium, silver and gold.

[0018] Preferably, when the number of layers of the metal film is 4, the metals of the bottom layer, interlayer 1, interlayer 2 and covering layer are respectively: titanium, gold, silver and gold.

[0019] Preferably, when the number of layers of the metal film is 4, the thickness of the bottom layer is 5-30 nm, the thickness of the interlayer 1 is 2-40 nm, the thickness of the interlayer 2 is 10-90 nm, and the thickness of the covering layer is 10-90 nm.

[0020] Further preferably, when the number of layers of the metal film is 4, the thickness of the bottom layer is 9 nm, the thickness of the interlayer 1 is 15 nm, the thickness of the interlayer 2 is 70 nm, and the thickness of the covering layer is 25 nm.

[0021] Preferably, when the number of layers of the metal film is 5, the metals of the bottom layer, interlayer 1, interlayer 2, interlayer 3 and covering layer are respectively: titanium, silver, gold, silver, gold.

[0022] Preferably, when the number of layers of the metal film is 6, the metals of the bottom layer, interlayer 1, interlayer 2, interlayer 3, interlayer 4 and covering layer are respectively: titanium, gold, silver, gold, silver and gold.

[0023] Preferably, when the number of layers of the metal film is 7, the metals of the bottom layer, interlayer 1, interlayer 2, interlayer 3, interlayer 4, interlayer 5 and covering layer are respectively: titanium, silver, gold, silver, gold, silver, gold.

[0024] Preferably, when the number of layers of the metal film is 8, the metals of the bottom layer, interlayer 1, interlayer 2, interlayer 3, interlayer 4, interlayer 5, interlayer 6 and covering layer are respectively: titanium, gold, silver, gold, silver, gold, silver, gold.

[0025] Preferably, when the number of layers of the metal film is 9, the metals of the bottom layer, interlayer 1, interlayer 2, interlayer 3, interlayer 4, interlayer 5, interlayer 6, interlayer 7 and covering layer are respectively: titanium, silver, gold, silver, gold, silver, gold, silver, and gold.

[0026] Preferably, when the number of layers of the metal film is 10, the metals of the bottom layer, interlayer 1, interlayer 2, interlayer 3, interlayer 4, interlayer 5, interlayer 6, interlayer 7, interlayer 8 and covering layer are respectively: titanium, gold, silver, gold, silver, gold, silver, gold, silver, and gold.

[0027] Preferably, when the number of layers of the metal film is 11, the metals of the bottom layer, interlayer 1, interlayer 2, interlayer 3, interlayer 4, interlayer 5, interlayer 6, interlayer 7, interlayer 8, interlayer 9 and covering layer are respectively: titanium, silver, gold, silver, gold, silver, gold, silver, gold, silver, and gold.

[0028] Preferably, the metal evaporation method is ion beam evaporation.

[0029] The preparation method of the novel supersurface SPR chip comprises the following steps:

[0030] (1) Using laser interference lithography and ion etching, a nanocup array structure is constructed on a silicon wafer to obtain a mold;

[0031] (2) placing the mold in a vacuum desiccator filled with hexylsilane for 12 h to make it hydrophobic;

[0032] (3) evenly coating the optical adhesive on the mold, placing a substrate material on top of the mold, curing it under ultraviolet light, and peeling it off to obtain a nanocup array on the substrate;

[0033] (4) Evaporate metal onto the surface of the nanocup array to obtain a metasurface SPR chip.

[0034] Preferably, the optical adhesive is Norland optical adhesive NOA-61.

[0035] The present invention is based on the design concept of making full use of different metal characteristics to achieve optimal performance. By accurately adjusting and controlling the type, number of layers and thickness of the metal, one or more metals are sequentially deposited on the surface of the nanocup array structure by ion beam evaporation to obtain a super surface SPR chip with a nanocup array. The SPR sensor of the present invention has higher sensitivity and stability. Regarding the number of layers of the metal film, when the number of layers of the metal film is greater than 3 layers, the intermediate interlayer of the present invention adopts the method of alternating deposition of gold film and silver film to prepare the obtained super surface SPR chip, which has the advantages of stability of gold film and high sensitivity of silver film; Regarding the thickness of the metal film, the present invention adjusts the total thickness of the metal film to 10~500 nm, wherein, when the number of layers of the metal film is 4, the thickness of the bottom metal film is adjusted to 5~30 nm, the thickness of the intermediate interlayer gold is adjusted to 2~40nm, the thickness of the intermediate interlayer silver is adjusted to 10~90 nm, and the thickness of the covering gold layer is adjusted to 10~90 nm. By combining different metals and different thicknesses, the super surface SPR chip can achieve higher detection sensitivity and stability. When the number of metal film layers is greater than 3, the present invention uses a gold-coated silver layer as a sensing layer, wherein the gold film can serve as a protective layer for the silver layer to prevent the silver layer from being oxidized or sulfided. In addition, the present invention also takes into account the influence of metal and metal oxide surface adsorbents, and by adjusting the combination and thickness of the metal film, the stability and sensitivity of the sensor can be significantly improved. The present invention not only solves the problem of insufficient stability and sensitivity in the prior art, but also provides an efficient, sensitive and stable solution for label-free affinity evaluation, which is suitable for the detection of various biomolecules such as protein-protein, protein-DNA, protein-small molecules, etc.

[0036] Compared with the prior art, the present invention is beneficial in that:

[0037] (1) The present invention makes full use of the characteristics of different metals and adopts different metals / metal oxides, different thicknesses and combinations to obtain a new type of metasurface SPR chip with higher response signals and sensitivity, and can detect biomarkers at lower concentrations; in addition, the combination of multiple metals / metal oxides can enhance the stability and reliability of the sensor and reduce the impact of organic material adsorption on chip performance;

[0038] (2) When the number of metal film layers is ≥3, the present invention uses gold as a covering layer, which can protect the silver coating through the gold film, solve the problem of silver oxidation or sulfidation sensitivity, and extend the service life of the sensor;

[0039] (3) The novel metasurface SPR chip of the present invention is easy to operate. Under the condition that the total thickness of the metal film is the same, titanium, metal oxide, silver, etc. are used to replace pure gold, which reduces the production cost of the chip and is conducive to large-scale production and application;

[0040] (4) The present invention uses a combination of multiple metals to improve the interface properties and enhance the stability and sensitivity of metal and metal oxide nanostructures;

[0041] (5) The present invention adopts a multilayer structure combination to achieve higher detection sensitivity and improved sensing performance, and can be applied to protein-protein, protein-DNA, and protein-small molecule molecular interaction detection. In addition, it can also be applied to label-free affinity evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The signal response results of the new metasurface chip with different thickness of interlayer gold layer, interlayer silver layer and covered gold layer to 5% sucrose solution;

[0043] Figure 2 a is the nanocup array structure diagram of the supersurface SPR chip; Figure 2 b is a schematic diagram of the periodic size of the nanocup array; Figure 2 c is a schematic diagram of the size of a single nanocup;

[0044] Figure 3 Schematic diagram of the structure of the metasurface SPR chip;

[0045] Figure 4 The sensitivity comparison results of the new supersurface SPR chips prepared in Examples 1 to 4 are shown in FIG. Figure 4 a is the differential spectrum of the transmission response signal of the novel metasurface SPR chip prepared in Example 1; Figure 4 b is the differential spectrum of the transmission response signal of the novel metasurface SPR chip prepared in Example 2; Figure 4 c is the differential spectrum of the transmission response signal of the novel metasurface SPR chip prepared in Example 3; Figure 4 d is the differential spectrum of the transmission response signal of the novel metasurface SPR chip prepared in Example 4; Figure 4 e is the dual-wavelength difference measured data result of the novel metasurface SPR chip prepared in Examples 1 to 4; Figure 4 f. Figure 4 g is the theoretical calculation result of dual wavelength difference of the novel metasurface SPR chip prepared in Examples 1 to 4;

[0046] Figure 5 is the result of Pro A and IgG affinity test; among them, Figure 5a is the real-time detection data and affinity calculation results of the affinity between Pro A and IgG in the novel supersurface SPR chip prepared in Example 1; Figure 5 b is the real-time detection data and affinity calculation results of the affinity between Pro A and IgG in the novel supersurface SPR chip prepared in Example 2; Figure 5 c is the real-time detection data and affinity calculation results of the affinity between Pro A and IgG in the novel supersurface SPR chip prepared in Example 3; Figure 5 d is the real-time detection data and affinity calculation results of the affinity of Pro A and IgG in the new supersurface SPR chip prepared in Example 4. DETAILED DESCRIPTION

[0047] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] The present invention provides a novel super-surface SPR chip, comprising a substrate, a nanocup array and a metal film, wherein the nanocup array is located on the substrate, and the metal film is evaporated on the surface of the nanocup array structure;

[0049] The number of layers of the metal film is 2 to 11;

[0050] When the number of layers of the metal film is 2, the metal film is divided into a bottom layer (i.e., the first layer) and a cover layer (i.e., the last layer);

[0051] When the number of layers of the metal film is 3 to 11, the metal film is divided into a bottom layer (i.e., the first layer), an interlayer and a cover layer (i.e., the last layer);

[0052] The metal of the bottom layer is selected from one of niobium pentoxide, tantalum pentoxide, zirconium oxide, hafnium dioxide and titanium;

[0053] The metal of the interlayer is selected from gold or silver;

[0054] The metal of the covering layer is gold.

[0055] In some examples, the material of the substrate includes, but is not limited to, PET (polyethylene terephthalate) material, PP (polypropylene) material, PE (polyethylene) material, and PVC (polyvinyl chloride) material.

[0056] In some examples, the thickness of the substrate is 10-1000 μm.

[0057] In some examples, the period of the nanocup array is 300-500 nm, and the upper diameter of a single nanocup is 150-300 nm, the lower diameter is 50-200 nm, and the height is 200-600 nm.

[0058] In some examples, the total thickness of the metal film is 10-500 nm.

[0059] Example 1

[0060] A novel super-surface SPR chip, the preparation steps are as follows:

[0061] (1) A nanocup array structure was constructed on a silicon wafer by laser interference lithography and ion etching to obtain a mold. The period of the nanocup array was 500 nm, and the upper diameter of a single nanocup was 200 nm, the lower diameter was 100 nm, and the height was 500 nm.

[0062] (2) placing the mold in a vacuum desiccator filled with hexylsilane for 12 h to make it hydrophobic;

[0063] (3) Evenly apply Norland optical adhesive NOA-61 on the mold, place a PET sheet on top of the mold, and irradiate with ultraviolet light (105 mW / cm 2 ) and then cured and peeled off to obtain a nanocup array on a PET substrate;

[0064] (4) Titanium and gold were sequentially deposited on the surface of the nanocup array by ion beam evaporation. The thickness of titanium and gold were 9 nm and 25 nm, respectively, to obtain a novel metasurface SPR chip.

[0065] Example 2

[0066] A novel super-surface SPR chip, the preparation steps are as follows:

[0067] (1) A nanocup array structure was constructed on a silicon wafer by laser interference lithography and ion etching to obtain a mold. The period of the nanocup array was 500 nm, and the upper diameter of a single nanocup was 200 nm, the lower diameter was 100 nm, and the height was 500 nm.

[0068] (2) placing the mold in a vacuum desiccator filled with hexylsilane for 12 h to make it hydrophobic;

[0069] (3) Evenly apply Norland optical adhesive NOA-61 on the mold, place a PET sheet on top of the mold, and irradiate with ultraviolet light (105 mW / cm 2 ) and then cured and peeled off to obtain a nanocup array on a PET substrate;

[0070] (4) Titanium, gold, and silver were sequentially deposited on the surface of the nanocup array by ion beam evaporation. The thickness of titanium, gold, and silver were 9 nm, 25 nm, and 70 nm, respectively, to obtain a novel metasurface SPR chip.

[0071] Example 3

[0072] A novel super-surface SPR chip, the preparation steps are as follows:

[0073] (1) A nanocup array structure was constructed on a silicon wafer by laser interference lithography and ion etching to obtain a mold. The period of the nanocup array was 500 nm, and the upper diameter of a single nanocup was 200 nm, the lower diameter was 100 nm, and the height was 500 nm.

[0074] (2) placing the mold in a vacuum desiccator filled with hexylsilane for 12 h to make it hydrophobic;

[0075] (3) Evenly apply Norland optical adhesive NOA-61 on the mold, place a PET sheet on top of the mold, and irradiate with ultraviolet light (105 mW / cm 2 ) and then cured and peeled off to obtain a nanocup array on a PET substrate;

[0076] (4) Titanium, gold, silver, and gold were sequentially deposited on the surface of the nanocup array by ion beam evaporation. The thicknesses of titanium, gold, silver, and gold were 9 nm, 25 nm, 70 nm, and 25 nm, respectively, to obtain a novel metasurface SPR chip.

[0077] Example 4

[0078] A novel super-surface SPR chip, the preparation steps are as follows:

[0079] (1) A nanocup array structure was constructed on a silicon wafer by laser interference lithography and ion etching to obtain a mold. The period of the nanocup array was 500 nm, and the upper diameter of a single nanocup was 200 nm, the lower diameter was 100 nm, and the height was 500 nm.

[0080] (2) placing the mold in a vacuum desiccator filled with hexylsilane for 12 h to make it hydrophobic;

[0081] (3) Evenly apply Norland optical adhesive NOA-61 on the mold, place a PET sheet on top of the mold, and irradiate with ultraviolet light (105 mW / cm 2 ) and then cured and peeled off to obtain a nanocup array on a PET substrate;

[0082] (4) Titanium, gold, silver, and gold were sequentially deposited on the surface of the nanocup array by ion beam evaporation. The thicknesses of titanium, silver, gold, silver, and gold were 9 nm, 70 nm, 25 nm, 70 nm, and 25 nm, respectively, to obtain a novel metasurface SPR chip.

[0083] Example 5

[0084] A novel super-surface SPR chip, the preparation steps are as follows:

[0085] (1) A nanocup array structure was constructed on a silicon wafer by laser interference lithography and ion etching to obtain a mold. The period of the nanocup array was 500 nm, and the upper diameter of a single nanocup was 200 nm, the lower diameter was 100 nm, and the height was 500 nm.

[0086] (2) placing the mold in a vacuum desiccator filled with hexylsilane for 12 h to make it hydrophobic;

[0087] (3) Evenly apply Norland optical adhesive NOA-61 on the mold, place a PET sheet on top of the mold, and irradiate with ultraviolet light (105 mW / cm 2 ) and then cured and peeled off to obtain a nanocup array on a PET substrate;

[0088] (4) Titanium, gold, silver, and gold are sequentially deposited on the surface of the nanocup array by ion beam evaporation to obtain a novel metasurface SPR chip.

[0089] The thickness of the bottom titanium layer in Example 5 was set to 9 nm, and the signal responses of the supersurface SPR chips (hereinafter referred to as chips) with different thicknesses of interlayer gold layer, interlayer silver layer, and gold layer were compared to the 5% sucrose solution. The specific operation was as follows:

[0090] Q1. Use ultrapure water to prepare RI-1.3403 (5%) sucrose aqueous solution;

[0091] Q2. Use a spray gun to add sucrose aqueous solution to the chip holes at 50 μL / well, and use a BioTEK continuous wavelength microplate reader for detection. The specific thicknesses of the interlayer gold layer, interlayer silver layer, and cover gold layer are shown in Table 1.

[0092] Table 1: Specific thickness of interlayer gold layer, interlayer silver layer and covering gold layer

[0093]

[0094] The results of the effects of different thicknesses of interlayer gold layer, interlayer silver layer, and covering gold layer on chip sensitivity are as follows Figure 1 shown. Figure 1The results show that the interlayer gold layer with a thickness of 15 nm produced the highest signal, indicating that 15 nm is the optimal thickness of the interlayer gold layer to maximize sensitivity; the interlayer silver layer with a thickness of 70 nm is associated with the highest signal among the tested thicknesses, indicating that 70 nm is the optimal thickness of the interlayer silver layer; for the overlay gold layer, it can be clearly seen from the figure that 25 nm is the optimal thickness, and the signal generated when the thickness is 25 nm is the highest compared to the thicknesses of 20 nm, 30 nm, and 40 nm. In summary, based on the signal response to the 5% sucrose solution and the detailed analysis of the metal layer thickness, the optimal deposition parameters for the chip were determined to be 9 nm Ti, 15 nm Au, 70 nm Ag, and 25 nm Au.

[0095] The supersurface SPR chip with deposition parameters of 9 nm Ti, 15 nm Au, 70 nm Ag, and 25 nm Au in Example 5 was characterized by AFM. Figure 2 a is the nanocup array structure diagram of the supersurface SPR chip. Figure 2 a It can be seen that the supersurface SPR chip has a uniform nanocup array structure; Figure 2 b is a schematic diagram of the periodic size of the nanocup array; Figure 2 c is a schematic diagram of the size of a single nanocup. Figure 3 is a schematic diagram of the structure of the metasurface SPR chip. Figure 3 As shown, chemical modification is performed on the basis of the super-surface SPR chip of the multilayer metal film, and then the ligand is fixed to detect the analyte, which can perform affinity detection and quantitative detection.

[0096] Using different concentrations of sucrose, the sensitivity of the new metasurface SPR chips prepared in Examples 1 to 4 was compared. Sucrose aqueous solutions with an RI range of 1.3332 to 1.4062 (percentage 0.15-50%, 2-fold gradient dilution) were prepared using water and compared with water (RI: 1.3330) and detected using a BioTEK continuous wavelength microplate reader. The specific steps are as follows:

[0097] (1) Use ultrapure water to prepare sucrose aqueous solutions of RI-1.3330 (water), RI-1.3332 (0.15%), RI-1.3335 (0.3%), RI-1.3339 (0.6%), RI-1.3348 (1.2%), RI-1.3367 (2.5%), RI-1.3403 (5%), RI-1.3476 (10%), and RI-1.3623 (20%).

[0098] (2) Use a spray gun to add 50 μL / well to the chip wells and use the BioTEK continuous wavelength microplate reader for detection. The test results are as follows: Figure 4 As shown; among them, Figure 4 a is the differential spectrum of the transmission response signal of the novel metasurface SPR chip prepared in Example 1; Figure 4 b is the differential spectrum of the transmission response signal of the novel metasurface SPR chip prepared in Example 2; Figure 4 c is the differential spectrum of the transmission response signal of the novel metasurface SPR chip prepared in Example 3; Figure 4 d is the differential spectrum of the transmission response signal of the novel metasurface SPR chip prepared in Example 4; Figure 4 e is the dual-wavelength difference measured data result of the novel metasurface SPR chip prepared in Examples 1 to 4; Figure 4 f. Figure 4 g is the theoretical calculation result of the dual wavelength difference of the new supersurface SPR chip prepared in Examples 1 to 4. Figure 4 From a to 4d, it can be seen that the gradient differentiation of the new supersurface SPR chip prepared in Example 3 is the most obvious and the change is the largest; Figure 4 It can be seen from e that the new metasurface SPR chip prepared in Example 3 has the highest response value; the above results show that the new metasurface SPR chip prepared in Example 3 has the highest sensitivity.

[0099] Using the chips prepared in Examples 1 to 4, affinity evaluation of Pro A and IgG was performed, and the specific steps were as follows:

[0100] S1. Use 75% ethanol and ultrapure water to clean the chip substrate alternately three times, and blow dry with nitrogen gas;

[0101] S2, add 10 mM thiohexanoic acid and 10 mM 11-mercaptoundecanoic acid in a volume ratio of 10:1 into the chip wells at 50 μL / well and incubate at 4 °C overnight;

[0102] S3, take out the chip, discard the mixed solution, wash the chip three times with ultrapure water, and blow dry with nitrogen;

[0103] S4, 0.4 M 1-ethyl-(3-dimethylaminopropyl) carbodiimide and 0.1 M N-hydroxysuccinimide were mixed in a volume ratio of 1:1, and 50 μL / well was added to the chip wells, incubated at 37 °C for 20 min, the chip was removed, the mixture was discarded, the chip was washed three times with ultrapure water, and dried with nitrogen gas;

[0104] S5. Dilute Protein A to 20 μg / mL with 50 mM 2-morpholineethanesulfonic acid, spot 2 μL in the center of the chip, and incubate at 4°C overnight.

[0105] S6, add 1% fish gelatin blocking solution and incubate at 37 °C for 1 h;

[0106] S7, take out the chip, shake off the excess blocking solution, wash the chip three times with ultrapure water, and blow dry with nitrogen;

[0107] S8. Use 10 mM PBS (Chinese name: phosphate buffer) with pH=7.4 to dilute the IgG monoclonal antibody to 66.7 nM, 33.3 nM, 16.7 nM, 8.3 nM, 4.2 nM, 2.1 nM, and 1 nM, and use the MetaSPR200 molecular interaction instrument for detection. The test results are as follows Figure 5 As shown, Figure 5 a~5d are respectively the real-time detection data and affinity calculation results of Pro A and IgG affinity in the chips of Examples 1~4. Figure 5 From a to 5d, it can be found that the affinity results of the four chips are almost the same, among which the dynamic detection response value (RU) of the chip prepared in Example 3 is the largest; this result shows that chips with different numbers of metal films will not affect the affinity of molecular interactions on the chip surface.

[0108] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A novel supersurface SPR chip, characterized in that: It includes a substrate, a nanocup array and a metal film, wherein the nanocup array is located on the substrate, and the metal film is evaporated on the surface of the nanocup array structure; The number of layers of the metal film is 2 to 11; When the number of layers of the metal film is 2, the metal film is divided into a bottom layer and a cover layer; When the number of layers of the metal film is 3 to 11, the metal film is divided into a bottom layer, an interlayer and a cover layer; The metal of the bottom layer is selected from one of niobium pentoxide, tantalum pentoxide, zirconium oxide, hafnium dioxide and titanium; The metal of the interlayer is selected from gold or silver; The metal of the covering layer is gold.

2. A novel supersurface SPR chip according to claim 1, characterized in that, The period of the nanocup array is 300-500 nm, and the upper diameter of a single nanocup is 150-300 nm, the lower diameter is 50-200 nm, and the height is 200-600 nm.

3. A novel supersurface SPR chip according to claim 1, characterized in that, When the number of layers of the metal film is 2, the metals of the bottom layer and the cover layer are titanium and gold respectively.

4. A novel supersurface SPR chip according to claim 1, characterized in that, When the number of layers of the metal film is 3, the metals of the bottom layer, the interlayer and the covering layer are respectively: titanium, silver and gold.

5. A novel supersurface SPR chip according to claim 1, characterized in that, When the number of layers of the metal film is 4, the metals of the bottom layer, interlayer 1, interlayer 2, and cover layer are respectively: titanium, gold, silver, and gold.

6. A novel supersurface SPR chip according to claim 1, characterized in that, When the number of layers of the metal film is 5, the metals of the bottom layer, interlayer 1, interlayer 2, interlayer 3 and covering layer are respectively: titanium, silver, gold, silver and gold.

7. A novel supersurface SPR chip according to claim 1, characterized in that, When the number of layers of the metal film is 6, the metals of the bottom layer, interlayer 1, interlayer 2, interlayer 3, interlayer 4 and covering layer are respectively: titanium, gold, silver, gold, silver and gold.

8. A novel supersurface SPR chip according to claim 1, characterized in that, When the number of layers of the metal film is 7, the metals of the bottom layer, interlayer 1, interlayer 2, interlayer 3, interlayer 4, interlayer 5 and covering layer are respectively: titanium, silver, gold, silver, gold, silver and gold.

9. A novel supersurface SPR chip according to claim 1, characterized in that, When the number of layers of the metal film is 8, the metals of the bottom layer, interlayer 1, interlayer 2, interlayer 3, interlayer 4, interlayer 5, interlayer 6 and covering layer are respectively: titanium, gold, silver, gold, silver, gold, silver and gold.

10. A method for preparing a novel supersurface SPR chip according to any one of claims 1 to 9, characterized in that: The following steps are involved: (1) Using laser interference lithography and ion etching, a nanocup array structure is constructed on a silicon wafer to obtain a mold; (2) placing the mold in a vacuum desiccator filled with hexylsilane for 12 h to make it hydrophobic; (3) evenly coating the optical adhesive on the mold, placing a substrate material on top of the mold, curing it under ultraviolet light, and peeling it off to obtain a nanocup array on the substrate; (4) Evaporate metal onto the surface of the nanocup array to obtain a metasurface SPR chip.

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