Interlayer surface plasma resonance biosensor as well as preparation method and application thereof

Abstract

The invention provides an interlayer surface plasmon resonance biosensor which comprises a patterned substrate, and detection areas which are arranged in an array and are formed by metal layers are formed on the surface of the patterned substrate; the spacing layer is formed in the detection area and comprises capture molecules; the nanometer adhesive tape is compounded on the surface of the patterned substrate, and a metal nanoparticle layer is arranged on one surface, facing the patterned substrate, of the nanometer adhesive tape. The invention further provides a preparation method and application of the interlayer surface plasma resonance biosensor. The nano double-sided adhesive tape is used as a transfer medium, and stable transfer of the metal nanoparticles from the substrate to the adhesive tape can be realized through two-step operation of'attaching-stripping ', so that the transfer difficulty of the metal nanoparticle layer is remarkably reduced, the operation is simplified, and the transfer efficiency is improved. And parameters such as the size, the morphology and the density of the nanoparticles in the sandwich structure can be accurately controlled, and the obtained metal nanoparticle layer is good in stability and can be stored for a long time.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and in particular to a sandwich surface plasmon resonance biosensor, its preparation method, and its application. Background Technology

[0002] Clinical immunological techniques based on protein qualitative or quantitative analysis are of great significance in many biomedical fields, including disease diagnosis, epidemiological research, immunological analysis, and molecular biology research. Surface plasmon resonance (SPR) is a physical instrument based on surface plasmon resonance technology, widely used for label-free real-time detection of intermolecular interactions. Sandwich-type surface plasmon resonance biosensors based on SPR technology exhibit a strong electromagnetic field enhancement effect on the interlayer medium, enabling highly sensitive detection of low concentrations of biomolecules and showing broad application prospects in point-of-care testing (POCT).

[0003] However, the mainstream method for constructing sandwich surface plasmon resonance biosensors is to drop-coat a metal nanoparticle solution onto a metal film surface or to use wet transfer. However, these methods are difficult to precisely control the distribution density and position of nanoparticles and cannot be prepared uniformly over a large area, thus limiting their large-scale application. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a sandwich surface plasmon resonance biosensor, its preparation method and its application. The preparation method provided by this application is simple, convenient and stable, and can prepare a large area uniformly.

[0005] This application provides a sandwich surface plasmon resonance biosensor, comprising:

[0006] A patterned substrate, wherein an array of detection areas formed of metal layers are formed on the surface of the patterned substrate;

[0007] A spacer layer is formed in the detection region, the spacer layer comprising trapping molecules;

[0008] A nano-adhesive tape is laminated onto the surface of the patterned substrate, wherein a layer of metal nanoparticles is disposed on the side of the nano-adhesive tape facing the patterned substrate.

[0009] In some specific implementations, the thickness of the spacer layer is 0.1 nm to 10 μm.

[0010] In some specific implementations, the non-detection areas of the patterned substrate surface have a hydrophobic surface.

[0011] In some specific implementations, the density of metal nanoparticles in the metal nanoparticle layer is 20 / μm.2 ~200000 / μm 2 The particle size of the metal nanoparticles ranges from 2 nm to 200 nm, and the metal nanoparticles are selected from gold nanoparticles, silver nanoparticles, copper nanoparticles, or aluminum nanoparticles.

[0012] In some specific implementations, the metal layer is selected from gold, silver, copper or aluminum; the thickness of the metal layer is greater than 10 nm.

[0013] This application also provides a method for fabricating a sandwich surface plasmon resonance biosensor, comprising the following steps:

[0014] Metal nanoparticles are attached to nano-adhesive tape to form a metal nanoparticle layer;

[0015] A patterned substrate is provided, wherein an array of detection regions formed of metal layers are formed on the surface of the patterned substrate, and spacer layers including trapping molecules are formed in the detection regions;

[0016] By combining the nano-tape with a patterned substrate, a sandwich layer of metal nanoparticles and a metal layer is formed, resulting in a plasmon resonance biosensor on the sandwich surface.

[0017] In some specific implementations, metal nanoparticles are attached to the nano-tape using the following method to form a metal nanoparticle layer:

[0018] a) Treat the substrate to form amino, thiol or charge modification on the substrate surface;

[0019] b) The modified substrate is immersed in a metal nanoparticle solution to form a metal nanoparticle film;

[0020] c) The nano-adhesive tape is attached to the side of the substrate with the metal nanoparticle film, soaked in deionized water, and after removing the substrate, a metal nanoparticle layer is formed on the surface of the nano-adhesive tape.

[0021] In some specific implementations, step a) specifically includes:

[0022] a1) Pre-treat the substrate;

[0023] a2) Form amino, thiol or charge modifications on the pretreated substrate.

[0024] In some specific implementations, step a1) specifically includes:

[0025] The substrate was ultrasonically cleaned sequentially with n-hexane, ethanol and water, and then heated in a piranha solution.

[0026] a2) specifically includes:

[0027] The pretreated substrate was modified with aminosilane compounds;

[0028] or,

[0029] The pretreated substrate was modified with thiol-based compounds;

[0030] or,

[0031] The pretreated substrate is charged with a charged substance.

[0032] This application also provides an application of the sandwich surface plasmon resonance biosensor described in the above technical solution or the sandwich surface plasmon resonance biosensor prepared by the preparation method described in the above technical solution in the detection of biomolecules.

[0033] This application provides a sandwich surface plasmon resonance biosensor, comprising: a patterned substrate with an array of detection regions formed by a metal layer on its surface; a spacer layer formed in the detection regions, the spacer layer including trapping molecules; and a nano-adhesive tape laminated to the surface of the patterned substrate, the side of the nano-adhesive tape facing the patterned substrate having a metal nanoparticle layer. This application uses a nano-double-sided adhesive tape as a transfer medium, achieving stable transfer of metal nanoparticles from the substrate to the tape through a simple two-step "attach-peel" operation, significantly reducing the difficulty of transferring the metal nanoparticle layer, simplifying the operation, and enabling precise control of parameters such as the size, morphology, and density of nanoparticles in the sandwich structure. The resulting metal nanoparticle layer exhibits good stability, can be stored long-term, and is readily available. Furthermore, this application uses a patterned substrate with an array of detection regions formed by a metal layer on its surface, allowing for flexible setting of sample types for different detection regions, achieving customized detection, supporting simultaneous detection of multiple indicators, realizing high-throughput detection, and requiring only 10μL to 15μL of sample volume for detection, enabling micro-volume detection. Therefore, the sandwich surface plasmon resonance biosensor provided in this application has the advantages of rapid detection results, convenient result interpretation, and good quantitative ability. It can realize label-free detection, high-throughput detection, and micro-detection, which greatly improves the practicality and operability of the sandwich surface plasmon resonance biosensor and provides a more convenient and efficient solution for disease screening and health monitoring. Attached Figure Description

[0034] Figure 1 A physical image of the surface structure of the sandwich surface plasmon resonance biosensor provided in the embodiments of this application;

[0035] Figure 2This is a schematic diagram of the stacked structure of the detection area of ​​the interlayer surface plasmon resonance biosensor provided in an embodiment of this application;

[0036] Figure 3 A photograph of a nanotape with a metal nanoparticle layer prepared for an embodiment of this application;

[0037] Figure 4 Photographs of hydrophilic and hydrophobic patterned gold substrates prepared for embodiments of this application;

[0038] Figure 5 Scanning electron microscope images of the stacked structure of the biosensor detection area provided in the embodiments of this application;

[0039] Figure 6 The graph shows the relationship between the thickness of the spacer layer and the reflectivity.

[0040] Figure 7 A curve showing the relationship between the thickness of the spacer layer and the grayscale value;

[0041] Figure 8 This is a curve showing the relationship between the concentration of hepatitis B surface antigen and grayscale values.

[0042] Figure 9 This is a schematic diagram of the detection process;

[0043] Figure 10 Photos taken with a microscope and a mobile phone;

[0044] Figure 11 The results of the sensitivity and specificity tests of the sandwich surface plasmon resonance biosensor provided in this application for viruses. Detailed Implementation

[0045] This invention provides a sandwich surface plasmon resonance biosensor, its preparation method, and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the same result. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0046] This application provides a sandwich surface plasmon resonance biosensor, comprising:

[0047] A patterned substrate, wherein an array of detection areas formed of metal layers are formed on the surface of the patterned substrate;

[0048] A spacer layer is formed in the detection region, the spacer layer comprising trapping molecules;

[0049] A nano-adhesive tape is laminated onto the surface of the patterned substrate, wherein a layer of metal nanoparticles is disposed on the side of the nano-adhesive tape facing the patterned substrate.

[0050] In the sandwich surface plasmon resonance biosensor provided in this application, there is a distance-dependent surface plasmon electromagnetic coupling between the metal nanoparticle layer and the metal layer. When the microscopic thickness of the spacer layer changes due to the reaction, the coupling strength between the metal nanoparticle layer and the metal layer also changes accordingly, causing the optical properties of the sandwich structure to change at visible light wavelengths. It can be identified and quantified by spectrometers, optical microscopes, naked eyes, mobile phone photography or portable fiber optic spectroscopy equipment, thereby realizing the visualization, multi-parameter, and high-throughput detection of biomolecules, such as proteins.

[0051] See Figure 1 and Figure 2 , Figure 1 The image shows the surface structure of the sandwich surface plasmon resonance biosensor provided in the embodiments of this application, where 11 is the detection area and 12 is the non-detection area. Figure 2 This is a schematic diagram of the stacked structure of the detection area of ​​the sandwich surface plasmon resonance biosensor provided in the embodiment of this application, wherein 21 is a nano-tape, 22 is a metal nanoparticle layer, 23 is a spacer layer, and 24 is a metal layer.

[0052] The sandwich surface plasmon resonance biosensor provided in this application includes a patterned substrate, on the surface of which an array of detection regions 11 formed by a metal layer are formed. In some specific implementations, multiple detection regions 11 form a detection unit, and the patterned substrate surface has an array of detection units, thereby achieving multi-throughput and multi-index detection. In some specific implementations, the non-detection region 12 on the surface of the patterned substrate has a hydrophobic surface, forming a hydrophilic-hydrophobic pattern with the detection regions 11 formed by the metal layer, realizing the self-confining function of droplets without the need for additional fencing devices.

[0053] In some specific implementations, the detection region 11 and the non-detection region 12 have different stacked structures. For example, the stacked structure of the detection region 11 sequentially includes a substrate (not shown in the figure), a metal layer 24, a spacer layer 23 containing trapped molecules, a metal nanoparticle layer 22, and a nanotape 21, while the stacked structure of the non-detection region 12 sequentially includes a substrate, an optional hydrophobic modification layer, a metal nanoparticle layer, and a nanotape. Those skilled in the art will understand that for the hydrophobic surface of the non-detection region, if the substrate is hydrophobic, such as a polydimethylsiloxane (PDMS) substrate, the non-detection region 12 does not need to be hydrophobically modified; if the substrate is hydrophilic, such as glass, the non-detection region 12 needs to be hydrophobically modified. This application does not impose any special restrictions on the specific method of hydrophobic modification, such as using fluorinating agents including 1H, 1H, 2H, 2H-perfluorooctyltrichlorosilane to form a hydrophobic surface.

[0054] In the sandwich surface plasmon resonance biosensor, nanotape 21 is used to fix metal nanoparticles, forming a metal nanoparticle layer. This application does not impose any special limitations on the nanotape; commercially available double-layer nanotape is sufficient. This application does not impose any special limitations on the method of fixing the metal nanoparticles on the nanotape; adhesion is sufficient. The metal nanoparticle layer 22 consists of metal nanoparticles capable of generating typical localized surface plasmon resonance phenomena, including but not limited to gold nanoparticles, silver nanoparticles, copper nanoparticles, or aluminum nanoparticles. This application does not impose any special limitations on the morphology of the metal nanoparticles, including but not limited to spherical, rod-shaped, star-shaped, cubic, or irregular shapes. This application does not impose any special limitations on the particle size of the metal nanoparticles; it can be 2nm–200nm, 5nm–190nm, 10nm–180nm, or 20nm–150nm, etc. This application does not impose any special limitations on the density of the metal nanoparticles on the nanotape; it can be 20 / μm. 2 ~200000 / μm 2 Or 50 / μm 2 ~150000 / μm 2 Or 100 / μm 2 ~120000 / μm 2 wait.

[0055] In a sandwich-type surface plasmon resonance biosensor, a spacer layer 23 is formed on the surface of the detection region 11. The spacer layer 23 includes capture molecules for the detection of biomolecules. This application does not impose any particular limitation on the capture molecules; they are molecules capable of binding to the analyte in the sample. For example, when the analyte is an antigen, the capture molecule is an antibody capable of specifically binding to it; when the analyte is an antibody, the capture molecule is an antigen capable of specifically binding to it. In some specific implementations, the capture molecules can be proteins, small molecules, polymers, aptamers, or nucleic acid molecules, determined according to the type of analyte. In some specific implementations, the spacer layer can be formed by liquid incubation, liquid drying, or printing with a microarray bioprinter. In some specific implementations, the thickness of the spacer layer is 0.1nm to 10μm, or 0.5nm to 8μm, or 0.10nm to 5μm, or 0.15nm to 3μm, or 0.2nm to 1μm, or 0.5nm to 500nm, or 1nm to 100nm, or 1.5nm to 50nm, or 2nm to 30nm.

[0056] In some specific implementations, the metal layer is a metal capable of electromagnetic coupling with the localized surface plasmon resonance of the metal nanoparticle layer, including but not limited to gold, silver, copper, or aluminum layers, with gold being preferred. In some specific implementations, the thickness of the metal layer is greater than 10 nm. This application does not impose any special limitations on the morphology of the metal layer, which can be a metal sheet, metal film, metal plating, etc.

[0057] The sandwich surface plasmon resonance biosensor also includes a substrate for supporting the metal layer 24. This application does not impose any special restrictions on the material of the substrate; any substrate commonly used by those skilled in the art is acceptable.

[0058] In some specific implementations, to increase the loading capacity and bioactivity of the trapped molecules in the spacer layer, the surface of at least one of the metal layer and the metal nanoparticle layer is physically or chemically modified, for example: polymer spin coating, polymer encapsulation, monolayer assembly, chemical grafting, atom transfer radical polymerization, plasma cleaning, and layer-by-layer self-assembly. This application does not impose any special limitations on the specific steps of each modification; they can be performed according to methods and parameters commonly used by those skilled in the art. In fact, the layer formed by this physical or chemical modification can be considered a spacer layer, which can be used to control the thickness of the spacer layer.

[0059] This application also provides a method for fabricating a sandwich surface plasmon resonance biosensor, comprising the following steps:

[0060] Metal nanoparticles are attached to nano-adhesive tape to form a metal nanoparticle layer;

[0061] A patterned substrate is provided, wherein an array of detection regions formed of metal layers are formed on the surface of the patterned substrate, and spacer layers including trapping molecules are formed in the detection regions;

[0062] By combining the nano-tape with a patterned substrate, a sandwich layer of metal nanoparticles and a metal layer is formed, resulting in a plasmon resonance biosensor on the sandwich surface.

[0063] This application uses nano-double-sided adhesive tape as a transfer medium. The stable transfer of metal nanoparticles from the substrate to the tape can be achieved through a simple two-step "attach-peel" operation, which significantly reduces the difficulty of transferring the metal nanoparticle layer, simplifies the operation, and can accurately control the size, morphology, density and other parameters of the nanoparticles in the sandwich structure. The obtained metal nanoparticle layer has good stability, can be stored for a long time, and can be used as needed.

[0064] In some specific implementations, metal nanoparticles are attached to the nano-tape using the following method to form a metal nanoparticle layer:

[0065] a) Treat the substrate to form amino, thiol or charge modification on the substrate surface;

[0066] b) The modified substrate is immersed in a metal nanoparticle solution to form a metal nanoparticle film;

[0067] c) The nano-adhesive tape is attached to the side of the substrate with the metal nanoparticle film, soaked in deionized water, and after removing the substrate, a metal nanoparticle layer is formed on the surface of the nano-adhesive tape.

[0068] Specifically, step a) includes:

[0069] a1) Pre-treat the substrate;

[0070] a2) Form amino, thiol or charge modifications on the pretreated substrate.

[0071] Specifically, step a1) includes:

[0072] The substrate was ultrasonically cleaned sequentially with hexane, ethanol and water, and then heated in a piranha solution.

[0073] This application can use glass, quartz, etc., as the substrate. First, it is ultrasonically cleaned with hexane, ethanol, and water, and then heated in a piranha solution. In some specific implementations, the ultrasonic cleaning time with hexane and ethanol is independently 3 min to 10 min, preferably 5 min; the ultrasonic cleaning with water is preferably performed twice, each time for independently 3 min to 10 min, preferably 5 min. In some specific implementations, the piranha solution is concentrated sulfuric acid and hydrogen peroxide in a volume ratio of 7:3; the heating temperature is 100℃ to 200℃, preferably 120℃ to 180℃; the time is 1 h to 3 h, preferably 1.5 h to 2.5 h. After the heating treatment, the substrate is cooled, ultrasonically cleaned with deionized water, and then soaked in ethanol for later use.

[0074] The pretreated substrate is modified with amino, thiol, or charge groups to enable it to bind metal nanoparticles and form a metal nanoparticle film. In some specific implementations, the amino modification can be performed as follows:

[0075] The pretreated substrate was modified with aminosilane compounds.

[0076] This application does not impose any special restrictions on the specific method of amino modification, and it can be carried out by means of soaking, vacuum heating gas phase reaction, etc.

[0077] In some specific implementations, a typical process of amino modification is as follows:

[0078] The pretreated substrate is dried with nitrogen and placed in a desiccator. An aminosilane solution such as (3-aminopropyl)trimethoxysilane (APTMS) is added, and a vacuum is drawn to bring the desiccator pressure to 0.5-5 mbar, preferably 1-3 mbar. The substrate is then placed in an oven at 50-100°C, preferably 60-80°C, and heated for 30 min to 2 hours, preferably 40 min to 1.5 h. The substrate is then removed and subjected to amino hydrolysis.

[0079] In some specific implementations, the thiol modification can be performed as follows:

[0080] The pretreated substrate was modified with thiol groups using thiol silane compounds.

[0081] After obtaining the amino or thiol-modified substrate, it is immersed in a metal nanoparticle solution, so that the metal nanoparticles are bonded to the substrate surface through electrostatic interaction or covalent bonding to form a metal nanoparticle film.

[0082] In some specific implementations, the charge modification can be performed in the following manner:

[0083] The pretreated substrate is charged with a charged substance.

[0084] In some specific implementations, the charge modification can be performed as follows: after pretreatment of the substrate by plasma treatment, it is immersed in a charged solution to form charge modification.

[0085] In a typical implementation, the charge modification is performed as follows:

[0086] The pretreated quartz substrate is dried with nitrogen and then placed in an air plasma cleaner for plasma treatment for 1 to 5 minutes, preferably 3 minutes. Then, it is immersed in a positively charged polyelectrolyte or a negatively charged solution for 10 to 20 minutes. After rinsing with water and drying, a substrate modified with positive or negative charges is obtained.

[0087] After obtaining a substrate modified with positive or negative charge, it is immersed in a metal nanoparticle solution, so that the metal nanoparticles are adsorbed onto the substrate surface through electrostatic interaction to form a metal nanoparticle film.

[0088] After obtaining a substrate with a metal nanoparticle film, a nano-adhesive tape is attached to the side of the substrate with the metal nanoparticle film. The substrate is then soaked in deionized water. After removing the substrate, a metal nanoparticle layer is formed on the surface of the nano-adhesive tape. The resulting nano-adhesive tape with the metal nanoparticle film can be stored long-term and used as needed.

[0089] This application uses a patterned substrate as the base, on the surface of which an array of detection regions formed by metal layers are formed. In some specific implementations, the non-detection regions of the patterned substrate have a hydrophobic surface. The structure of the patterned substrate is as described above, and will not be repeated here.

[0090] In some specific implementations, a typical fabrication process for the patterned substrate is as follows:

[0091] A metal layer is formed on the surface after the substrate is pretreated;

[0092] The metal layer is patterned using photoresist to form a detection area composed of the metal layer;

[0093] The non-detection area is fluorinated to form a hydrophobic surface;

[0094] After removing the photoresist, a patterned substrate is obtained.

[0095] Specifically, a typical fabrication process for the patterned substrate is as follows:

[0096] After pretreatment of the substrate, a chromium adhesion layer of 1-5 nm and a metal layer of more than 70 nm are formed on its surface by vacuum thermal evaporation deposition.

[0097] The metal layer is exposed by photoresist, developed with 0.5% NaOH, and the metal layer and chromium adhesion layer in the non-detection area are etched away to form the detection area composed of the metal layer;

[0098] The non-detection area is subjected to plasma treatment and then immersed in a fluorinated silane solution for fluorination to form a hydrophobic surface.

[0099] After removing the photoresist with ethanol, a patterned substrate is obtained.

[0100] In some specific implementations, the patterned substrate is prepared according to the following method:

[0101] The substrate is pretreated and then fluorinated.

[0102] A metal layer is formed on the fluorinated substrate;

[0103] The metal layer is patterned using photoresist to form a detection area composed of the metal layer;

[0104] The patterned substrate is obtained by transferring the pattern onto a hydrophobic material.

[0105] In some specific implementations, the pretreatment, fluorination treatment, metal layer formation, and patterning treatment can all refer to the methods described above, and will not be repeated here. A specific process of transfer printing is as follows:

[0106] A thin layer of UV-curable adhesive, such as Norland #61, is spin-coated onto the patterned substrate surface. Then, a hydrophobic material, such as polydimethylsiloxane (PDMS), is plasma-treated and bonded together with the front side facing down and free of air bubbles. The substrate is then cured under UV light for 20 minutes, peeled off, and flipped to obtain the patterned hydrophobic substrate.

[0107] After obtaining the patterned substrate, a spacer layer comprising the captured molecules is formed in its detection region. As mentioned above, the spacer layer can be formed by methods such as liquid incubation, liquid drying, or printing with a microarray bioprinter. In this application, a typical method for forming the spacer layer is as follows:

[0108] A solution containing the captured molecules is spotted onto the detection area of ​​a patterned substrate and dried at room temperature for 1-4 hours to form a spacer layer. After obtaining a nanotape with a metal nanoparticle layer and a patterned substrate with a spacer layer, the nanotape and the patterned substrate are composited to form a sandwich between the metal nanoparticle layer and the metal layer, resulting in a sandwich-surface plasmon resonance biosensor. This application does not impose any particular limitation on the composite method; simply attaching the nanotape flatly to the surface of the patterned gold substrate is sufficient.

[0109] Specifically, this application can reserve a sample application channel on the surface of the plasmon resonance biosensor on the sandwich surface. After adding trapping molecules to form a spacer layer, the nano-adhesive tape with a metal nanoparticle layer and the patterned substrate with the spacer layer are combined. During detection, the sample to be tested is added to the detection area through the sample application channel, and detection is performed after incubation. A typical method is as follows:

[0110] After the plasmon resonance biosensor on the sandwich surface was equilibrated at room temperature, it was sealed with a sealing agent, cleaned with phosphate buffer, rinsed with deionized water, and dried with nitrogen.

[0111] After adding the sample to be tested to the detection area, incubate it, then wash it with phosphate buffer, wash it with deionized water, dry it with nitrogen, and then perform the test.

[0112] In some specific implementations, after the plasmon resonance biosensor on the sandwich surface is equilibrated at room temperature, it is blocked with bovine serum albumin for 30 to 90 minutes, preferably 60 minutes. After blocking, it is washed with phosphate buffer, rinsed with deionized water, and dried with nitrogen. The sample to be tested is added to the detection area and incubated for 10 to 60 minutes. Then it is washed with phosphate buffer, rinsed with deionized water, dried with nitrogen, and then detected.

[0113] In some specific implementations, this application can also add capturing molecules and then incubate the sample to be tested to form a spacer layer. Then, the nano-tape with the metal nanoparticle layer and the patterned substrate with the spacer layer are combined to perform detection. A typical processing method is as follows:

[0114] The solution containing the captured molecules was spotted onto the detection area of ​​the patterned substrate, dried at room temperature for 1 to 4 hours, and stored under vacuum at 0 to 10°C.

[0115] After equilibrating the patterned substrate at room temperature, it was blocked with a blocking agent, washed with phosphate buffer, rinsed with deionized water, and dried with nitrogen.

[0116] After adding the sample to be tested to the detection area, incubate it, then wash it with phosphate buffer, wash it with deionized water, and blow it dry with nitrogen to form a sealing layer.

[0117] Then, the nano-tape with the metal nanoparticle layer and the patterned substrate with the spacer layer are combined and tested.

[0118] In some specific implementations, after the patterned substrate is equilibrated at room temperature, it is blocked with bovine serum albumin for 30 to 90 minutes, preferably 60 minutes. After blocking, it is washed with phosphate buffer and then rinsed with deionized water and dried with nitrogen. The sample to be tested is added to the detection area and incubated for 10 to 60 minutes. Then it is washed with phosphate buffer and deionized water, dried with nitrogen, and then combined with nanotape with a metal nanoparticle layer.

[0119] The sandwich surface plasmon resonance biosensor provided in this application relies on a thickness detection mechanism. Microscopic changes in the spacer layer will cause changes in the coupling strength between the metal nanoparticle layer and the metal layer, which will change the optical properties of the sandwich structure in the visible light band. The optical characterization and signal of the sandwich surface plasmon resonance biosensor can be read using the naked eye or a smartphone, and the signal is easy to read.

[0120] In this application, after taking a photo using a smartphone, the signal value is obtained by the difference between the grayscale signal of the sample points in the photo and the blank area. In some specific implementations, smartphone photography is combined with machine learning. Machine learning algorithms can directly extract the image grayscale values ​​to obtain the corresponding signal value. For example, a convolutional neural network can be used to train on a large number of photos, establishing a relationship between existing photos and grayscale values. Then, by directly recognizing the photo, the signal value can be directly output. The algorithm can be made into a mobile application program that allows direct image upload and output of results.

[0121] This application also provides the application of the sandwich surface plasmon resonance biosensor described in the above technical solution or the sandwich surface plasmon resonance biosensor prepared by the preparation method described in the above technical solution in the detection of biomolecules.

[0122] This application utilizes nano-double-sided adhesive tape as the transfer medium, achieving stable transfer of metal nanoparticles from the substrate to the tape through a simple two-step "attach-peel" operation. This significantly reduces the difficulty of transferring the metal nanoparticle layer, simplifies the operation, and allows for precise control of parameters such as the size, morphology, and density of nanoparticles in the sandwich structure. The resulting metal nanoparticle layer exhibits good stability, can be stored long-term, and is readily available for use. Furthermore, this application employs a patterned substrate with an array of detection regions formed by the metal layer on its surface. This allows for flexible setting of sample types for different detection regions, enabling customized detection. It also supports simultaneous detection of multiple indicators, achieving high-throughput detection, and requires only 10μL to 15μL of sample volume for detection, enabling micro-volume detection. Therefore, the sandwich surface plasmon resonance biosensor provided by this application has advantages such as rapid detection results, convenient result interpretation, and good quantitative capabilities. It can achieve label-free detection, high-throughput detection, and micro-volume detection, significantly improving the practicality and operability of the sandwich plasmon resonance biosensor and providing a more convenient and efficient solution for disease screening and health monitoring.

[0123] The present application will be further described below with reference to the embodiments.

[0124] Example 1

[0125] 1. Transfer of metal nanoparticles using adhesive tape:

[0126] (1) Quartz substrate pretreatment:

[0127] The quartz substrate was ultrasonically cleaned with hexane and ethanol for 5 min in sequence, and then ultrasonically cleaned twice with deionized water for 5 min each time. The pretreated quartz substrate was heated at 140℃ for 2 h in piranha solution (concentrated sulfuric acid: hydrogen peroxide = 7:3), cooled down, ultrasonically cleaned with deionized water, and then soaked in ethanol for later use.

[0128] (2) Forming a layer of metal nanoparticles on the surface of a quartz substrate:

[0129] The pretreated quartz substrate was dried with nitrogen and placed in a desiccator. 40 μL of a 97% (3-aminopropyl)trimethoxysilane (APTMS) solution was added, and the desiccator was evacuated to a pressure of 1 mbar. The substrate was then heated in a 70°C oven for 1 hour. After removal and 2 hours of amino hydrolysis, the substrate was directly immersed in 20 mL of the stock gold nanoparticle solution. After 24 hours of immersion, the solution was aspirated, and another 20 mL of gold nanoparticle solution was added to continue immersion, ensuring a gold nanoparticle density of 500–1000 particles / μm on the quartz substrate surface. 2 Finally, remove it, rinse it with water, and dry it with nitrogen before use.

[0130] (3) Tape transfer steps:

[0131] Apply the nano-double-sided adhesive tape flatly to the surface of a quartz substrate containing adsorbed metal nanoparticles. Immerse in deionized water for 5-10 minutes, then peel off the tape from the substrate, transferring the metal nanoparticles to one side of the tape. Dry with nitrogen gas for later use. See also Figure 3 , Figure 3 Photograph of a nanotape with a metal nanoparticle layer prepared for an embodiment of this application.

[0132] 2. Preparation of hydrophilic and hydrophobic patterned gold substrates

[0133] (1) Glass slide pretreatment

[0134] The glass substrate was ultrasonically cleaned with hexane and ethanol for 5 min in sequence, and then ultrasonically cleaned twice with deionized water for 5 min each time. The pretreated quartz substrate was heated at 140℃ for 2 h in piranha solution (concentrated sulfuric acid: hydrogen peroxide = 7:3), cooled down, ultrasonically cleaned with deionized water, and then soaked in ethanol for later use.

[0135] (2) Vacuum thermal evaporation deposition

[0136] A 3 nm chromium adhesion layer and a 70 nm gold layer were formed on the glass slide using a vacuum thermal evaporation method.

[0137] (3) Photolithographic patterning:

[0138] Positive photoresist is spin-coated onto the gold layer and dried, then exposed to the mask (using an ultraviolet lithography machine), and developed with a 0.5% NaOH solution to form a pattern.

[0139] (4) Wet etching: Gold and chromium layers are removed by sequentially treating with gold etchant and chromium etchant, rinsed with deionized water, and dried with nitrogen.

[0140] (5) Selective fluorination:

[0141] The glass slides were cleaned using an air plasma cleaner (Plasma). After 10 minutes, the glass slides were immersed in a hexane solution of 0.05% 1H,1H,2H,2H-perfluorooctyltrichlorosilane to treat the glass regions and form hydrophobic regions.

[0142] (6) Post-processing:

[0143] The protective photoresist was removed with ethanol, and then dried with nitrogen to obtain a hydrophilic-hydrophobic patterned gold substrate. See [link to documentation]. Figure 4 , Figure 4 Photograph of the hydrophilic and hydrophobic patterned gold substrate prepared for the embodiments of this application.

[0144] 3. Biomolecular detection

[0145] (1) Substrate pretreatment:

[0146] A concentration of 50–100 μg / mL -1 Capture molecule solutions, such as hepatitis B surface antibody, SARS-CoV-2 Alpha subtype antigen (AlphaAg), cytomegalovirus antigen (CMVAg), mycoplasma pneumoniae antigen (M.PAg), human herpes simplex virus type 1 antigen (HSV1 Ag), and SARS-CoV-2 BQ1.1 subtype antigen (BQ1.1 Ag), were spotted onto the detection area of ​​the hydrophilic-hydrophobic patterned gold substrate obtained in step 2, with a spotting volume of 0.3 μL. The samples were dried at room temperature for 2 h and then stored under vacuum at 4 °C.

[0147] (2) Detection operation:

[0148] After equilibration at room temperature, add 50 mg / mL bovine serum albumin to the spotting area for blocking for 1 h, wash with phosphate buffer (PBST / PBS) for 1 min, rinse with deionized water and dry with nitrogen, then add 10-15 μL of the sample to be tested, incubate for 30 min, then wash with PBST / PBS buffer for 1 min, rinse with deionized water and dry with nitrogen.

[0149] 4. Sensor assembly and testing

[0150] (1) Construction of the sandwich structure:

[0151] The side of the double-sided nano-adhesive tape obtained in step 1, with the metal nanoparticles adhered to it, was smoothly attached to the reacted gold substrate obtained in step 3 to obtain a sandwich-type surface plasmon resonance biosensor. The stacked structure of the detection area of ​​the sandwich-type surface plasmon resonance biosensor was scanned by electron microscopy; the results are shown in [reference needed]. Figure 5 , Figure 5 The image shows a scanning electron microscope (SEM) image of the stacked structure of the biosensor detection area provided in the embodiments of this application, wherein 51 is an adhesive tape, 52 is a metal nanoparticle, 53 is a spacer layer, 54 is a metal layer, and 55 is a substrate.

[0152] (2) Detection:

[0153] The spectral reflectance and grayscale value of the plasmon resonance biosensor on the interlayer surface were detected using a fiber optic spectrometer and an optical microscope; or a smartphone was used to photograph the plasmon resonance biosensor on the interlayer surface.

[0154] Example 2

[0155] The difference from Example 1 is that the hydrophilic-hydrophobic patterned gold substrate in step 2 is prepared according to the following method, while all other steps and parameters are the same:

[0156] (1) Silicon wafer pretreatment

[0157] The silicon wafer substrate was ultrasonically cleaned with hexane and ethanol for 5 minutes in sequence, and then ultrasonically cleaned twice with deionized water for 5 minutes each time. The pretreated silicon wafer substrate was heated at 140°C for 2 hours in a piranha solution (concentrated sulfuric acid: hydrogen peroxide = 7:3). After cooling, it was ultrasonically cleaned with deionized water and then soaked in ethanol for later use.

[0158] (2) Vacuum thermal evaporation deposition

[0159] After cleaning the pretreated silicon wafers with an air plasma machine for 10 minutes, they were immersed in a hexane solution of 0.05% 1H,1H,2H,2H-perfluorooctyltrichlorosilane and reacted for 10 minutes. The wafers were then removed, cleaned with ethanol, and dried with nitrogen for later use.

[0160] A 70nm gold layer was formed on the silicon wafer using a vacuum thermal evaporation method.

[0161] (3) Photolithographic patterning:

[0162] Positive photoresist is spin-coated onto the gold layer and dried, then exposed to the mask (using an ultraviolet lithography machine), and developed with a 0.5% NaOH solution to form a pattern.

[0163] The obtained silicon wafer is immersed in a gold etching solution to form a detection area and a non-detection area.

[0164] (4) PDMS transfer:

[0165] Spin-coat a thin layer of UV-curable adhesive (Norland #61) onto the patterned substrate surface, and treat the cured PDMS with Plasma (3 minutes). After bonding with the front side down and without air bubbles, cure under UV light for 20 minutes, peel off and flip to obtain the patterned PDMS substrate.

[0166] Experimental Example 1

[0167] Different concentrations of toluene solutions of polystyrene were spin-coated onto a gold substrate to form polymer layers with thicknesses of 3.3 nm, 6.8 nm, 10.0 nm, 14.6 nm, 18.3 nm, and 24.2 nm. The spectral reflectance and grayscale values ​​of the plasmon resonance biosensor on the interlayer surface were detected using fiber optic spectroscopy and optical microscopy, respectively. Curves showing the relationship between interlayer thickness and reflectance, and between interlayer thickness and grayscale values, were established. The results are detailed below. Figure 6 and Figure 7 , Figure 6 This is a graph showing the relationship between the thickness of the spacer layer and reflectivity. Figure 7 This is a curve showing the relationship between the thickness of the spacer layer and the grayscale value. Figure 6 and Figure 7 It can be seen that the interlayer surface plasmon resonance biosensor has a highly sensitive thickness response, with a spectral reflectance sensitivity of 0.541% nm.-1 The grayscale sensitivity reaches 2.390nm. -1 .

[0168] Experimental Example 2

[0169] In the gold detection area, hepatitis B surface antibody solution was spotted as a capture molecule, and 10 μg mL of the solution was added. -1 1 μg / mL -1 100ng mL -1 10ng mL -1 1 ng / mL -1 and 100 pg mL -1 The grayscale value of the plasmon resonance biosensor on the surface of the interlayer was detected using an optical microscope to obtain the HBsAg standard protein solution. A curve of hepatitis B surface antigen concentration versus grayscale value was established. The results are shown in [reference needed]. Figure 8 , Figure 8 This is a curve showing the hepatitis B surface antigen concentration versus grayscale value. Figure 8 It is evident that the interlayer surface plasmon resonance biosensor possesses high detection sensitivity, with a detection limit of 0.64 ng / mL for hepatitis B surface antigen. -1 .

[0170] Experimental Example 3

[0171] The difference from Example 2 is that different detection areas were coated with hepatitis B surface antibody (HBsAb), SARS-CoV-2 Alpha subtype antigen (AlphaAg), cytomegalovirus antigen (CMV Ag), mycoplasma pneumoniae antigen (M.PAg), human herpes simplex virus type 1 antigen (HSV1 Ag), and SARS-CoV-2 BQ1.1 subtype antigen (BQ1.1 Ag) as capture molecules, such as... Figure 9 As shown, Figure 9 This is a schematic diagram showing the positions of different captured molecules in a single detection unit, with the detection area in the middle serving as a blank control. The same serum sample was added to the same detection unit. This serum sample may contain one or more of the following: hepatitis B surface antigen, SARS-CoV-2 Alpha subtype antibody, cytomegalovirus antibody, mycoplasma pneumoniae antibody, human herpes simplex virus type 1 antibody, and SARS-CoV-2 BQ1.1 subtype antibody. The plasma resonance biosensor on the interlayer surface was photographed using an optical microscope and a mobile phone, respectively. The results are shown in [reference needed]. Figure 10 , Figure 10 Images taken with a microscope and a mobile phone, showing the trapped molecules in each detection area and... Figure 9The results are completely consistent. (a) and (b) are microscopic and mobile phone photos of the first serum sample, respectively. As shown, this serum is positive for SARS-CoV-2 Alpha subtype antibody, cytomegalovirus antibody, human herpes simplex virus type 1 antibody, and SARS-CoV-2 BQ1.1 subtype antibody. (c) and (d) are microscopic and mobile phone photos of the second serum sample, respectively. As shown, this serum is positive for Mycoplasma pneumoniae antibody, SARS-CoV-2 Alpha subtype antibody, cytomegalovirus antibody, human herpes simplex virus type 1 antibody, and SARS-CoV-2 BQ1.1 subtype antibody. (e) and (f) are microscopic and mobile phone photos of the third serum sample, respectively. As shown, this serum is positive for hepatitis B surface antigen, Mycoplasma pneumoniae antibody, and human herpes simplex virus type 1 antibody. (g) and (h) are microscopic and mobile phone photos of the fourth serum sample, respectively. As shown, this serum is positive for SARS-CoV-2 Alpha subtype antibody and human herpes simplex virus type 1 antibody. In the microscope images, darker colors indicate positive results, and lighter colors indicate negative results; in the mobile phone photos, darker red colors indicate positive results, and lighter red colors indicate negative results.

[0172] 255 serum samples were tested, and each sample was tested for the above six antigens and antibodies. The sensitivity and specificity of each antigen and antibody detection were obtained. Microscopic image grayscale analysis was used, and the positive or negative results of the clinical outcome were used as the standard. The results are shown in [link to results]. Figure 11 , Figure 11 The results show the sensitivity and specificity of the sandwich surface plasmon resonance biosensor provided in this application for viral biomarkers. Figure 11 It is evident that the interlayer surface plasmon resonance biosensor provided in this application has high sensitivity and good specificity for the detection of viral biomarkers.

[0173] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A sandwich surface plasmon resonance biosensor, characterized in that, include: A patterned substrate, wherein an array of detection areas formed of metal layers are formed on the surface of the patterned substrate; A spacer layer is formed in the detection region, the spacer layer comprising trapping molecules; A nano-adhesive tape is laminated onto the surface of the patterned substrate, wherein a layer of metal nanoparticles is disposed on the side of the nano-adhesive tape facing the patterned substrate.

2. The sandwich surface plasmon resonance biosensor according to claim 1, characterized in that, The thickness of the spacer layer is 0.1 nm to 10 μm.

3. The sandwich surface plasmon resonance biosensor according to claim 1 or 2, characterized in that, The non-detection area of ​​the patterned substrate surface has a hydrophobic surface.

4. The sandwich surface plasmon resonance biosensor according to claim 3, characterized in that, The density of the metal nanoparticles in the metal nanoparticle layer is 20 / μm. 2 ~200000 / μm 2 The particle size of the metal nanoparticles ranges from 2 nm to 200 nm, and the metal nanoparticles are selected from gold nanoparticles, silver nanoparticles, copper nanoparticles, or aluminum nanoparticles.

5. The sandwich surface plasmon resonance biosensor according to claim 3, characterized in that, The metal layer is selected from gold, silver, copper or aluminum; the thickness of the metal layer is greater than 10 nm.

6. A method for fabricating a sandwich surface plasmon resonance biosensor, characterized in that, Includes the following steps: Metal nanoparticles are attached to nano-adhesive tape to form a metal nanoparticle layer; A patterned substrate is provided, wherein an array of detection regions formed of metal layers are formed on the surface of the patterned substrate, and spacer layers including trapping molecules are formed in the detection regions; By combining the nano-tape with a patterned substrate, a sandwich layer of metal nanoparticles and a metal layer is formed, resulting in a plasmon resonance biosensor on the sandwich surface.

7. The preparation method according to claim 6, characterized in that, Metal nanoparticles are attached to nano-adhesive tape to form a metal nanoparticle layer using the following method: a) Treat the substrate to form amino, thiol or charge modification on the substrate surface; b) The modified substrate is immersed in a metal nanoparticle solution to form a metal nanoparticle film; c) The nano-adhesive tape is attached to the side of the substrate with the metal nanoparticle film, soaked in deionized water, and after removing the substrate, a metal nanoparticle layer is formed on the surface of the nano-adhesive tape.

8. The preparation method according to claim 7, characterized in that, Step a) specifically includes: a1) Pre-treat the substrate; a2) Form amino, thiol or charge modifications on the pretreated substrate.

9. The preparation method according to claim 7, characterized in that, Step a1) specifically includes: The substrate was ultrasonically cleaned sequentially with n-hexane, ethanol and water, and then heated in a piranha solution. a2) specifically includes: The pretreated substrate was modified with aminosilane compounds; or, The pretreated substrate was modified with thiol-based compounds; or, The pretreated substrate is charged with a charged substance.

10. The application of the sandwich surface plasmon resonance biosensor according to any one of claims 1 to 5 or the sandwich surface plasmon resonance biosensor prepared by the preparation method according to any one of claims 6 to 9 in the detection of biomolecules.

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