An SPR sensor interface for detecting SARS-CoV-2 antigen and its detection method

Abstract

The present invention provides an SPR sensor interface for detecting SARS-CoV-2 antigen and a detection method thereof, belonging to the technical field of SPR sensors. In the present invention, Cu3(PO4)2-BSA-GO nanoflowers are used to immobilize the ACE2 protein probe. The amide group and carboxyl group of the ACE2 protein probe will coordinate with the copper metal ions of the nanoflowers. In addition, the biological probe also binds to Cu 2+ and plays an inducing role, further enabling the Cu3(PO4)2-BSA-GO nanoflowers to tightly "adhere" to the ACE2 protein probe; the ACE2 protein can specifically bind to the SARS-CoV-2 antigen, binding the SARS-CoV-2 antigen to the surface of the Cu3(PO4)2-BSA-GO nanoflower layer, causing a change in the SPR angle of the SPR sensor interface, thereby realizing the detection of the content of SARS-CoV-2 antigen in the sample to be tested.

Description

Technical Field

[0001] The present invention relates to the technical field of SPR sensors, and in particular to an SPR sensor interface for detecting SARS-CoV-2 antigens and a detection method thereof. Background Art

[0002] The two mainstream methods for detecting the new coronavirus are molecular diagnostic technology based on polymerase chain reaction (PCR), nucleic acid hybridization and second-generation sequencing technology, and immunodiagnostic technology that detects antigens or antibodies produced in the body after patients are exposed to the new coronavirus. The former takes a long time to output results, and the latter has low accuracy, neither of which can match the needs of detecting the new coronavirus in aerosols in the air in large spaces. Therefore, it is necessary to find a real-time, fast, stable, and accurate detection method that can handle a large number of accumulated samples.

[0003] Surface plasmon resonance (SPR) detection technology is a label-free detection method used for real-time, highly sensitive, and measurable detection between biological samples, which meets the needs of aerosol detection of new coronavirus in public space environments. However, the traditional SPR sensor interface is usually used to fix the designed biological probe by chemical bonding through Au-S bonds, which is prone to shedding. Summary of the invention

[0004] In view of this, the present invention aims to provide an SPR sensor interface for detecting SARS-CoV-2 antigens and a detection method thereof. The SPR sensor interface provided by the present invention can effectively fix biological probes and realize accurate detection of SARS-CoV-2 antigens.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides an SPR sensor interface for detecting SARS-CoV-2 antigens, comprising a high refractive index glass, an indium tin oxide layer, an amorphous silicon layer, a gold layer and a Cu layer stacked in sequence. 3 (PO 4 ) 2 -BSA-GO nanoflower layer, the Cu 3 (PO 4 ) 2 -BSA-GO nanoflower layer includes Cu coated with ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflowers;

[0007] The Cu 3 (PO 4 ) 2-BSA-GO nanoflowers were obtained by precipitation reaction of copper sulfate, bovine serum albumin and graphene oxide in phosphate buffer;

[0008] The refractive index of the high refractive index glass is 1.72 to 1.82;

[0009] The Cu 3 (PO 4 ) 2 -The thickness of the BSA-GO nanoflower layer is 20 to 40 nm.

[0010] Preferably, the thickness of the high refractive index glass is 0.75 to 1 mm;

[0011] The thickness of the indium tin oxide layer is 60 to 100 nm;

[0012] The thickness of the amorphous silicon layer is 80 to 120 nm;

[0013] The thickness of the gold layer is 30-50 nm.

[0014] Preferably, the mass ratio of copper sulfate, bovine serum albumin and graphene oxide is 16:25-35:12.5-17.5;

[0015] The temperature of the precipitation reaction is 20-37° C. and the time is 20-30 hours.

[0016] Preferably, the ACE2 protein and Cu 3 (PO 4 ) 2 -The mass ratio of BSA-GO nanoflowers is 1:6~10.

[0017] The present invention provides a method for preparing the above-mentioned SPR sensor interface for detecting SARS-CoV-2 antigens, comprising the following steps:

[0018] First magnetron sputtering indium tin oxide onto the surface of the high refractive index glass to form an indium tin oxide layer;

[0019] performing a second magnetron sputtering of amorphous silicon onto the surface of the indium tin oxide layer to form an amorphous silicon layer;

[0020] magnetron sputtering gold onto the surface of the amorphous silicon layer to form a gold layer;

[0021] ACE2 protein, Cu 3 (PO 4 ) 2 -BSA-GO nanoflowers were mixed with a buffer solution and incubated for the first time to obtain Cu coated with ACE2 protein probes. 3 (PO 4 ) 2-BSA-GO nanoflowers;

[0022] The Cu coated with ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflowers were mixed with bovine serum albumin and incubated for the second time to obtain closed Cu coated with ACE2 protein probes. 3 (PO 4 ) 2 -BSA-GO nanoflowers;

[0023] The blocked Cu coated with ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflowers are deposited on the surface of the gold layer to obtain an SPR sensor interface for detecting SARS-CoV-2 antigens.

[0024] Preferably, the gas used in the first magnetron sputtering is helium, the sputtering pressure is 1-1.4 Pa, the sputtering power is 120-180 W, the sputtering time is 3-5 min, and the sputtering vacuum is 6×10 -4 Pa; the distance between the sputtering target source and the high refractive index glass is 8 to 12 cm.

[0025] Preferably, the gas used in the second magnetron sputtering is helium, the sputtering pressure is 2-3 Pa, the sputtering power is 150-250 W, the sputtering time is 0.8-1.2 h, and the sputtering vacuum is 6×10 -4 Pa; the distance between the sputtering target source and the indium tin oxide layer is 8 to 12 cm.

[0026] Preferably, the gas used in the third magnetron sputtering is helium, the sputtering pressure is 2.5-3.5 Pa, the sputtering power is 240-260 W, the sputtering time is 30-50 s, and the sputtering vacuum is 6×10 -4 Pa; the distance between the sputtering target source and the amorphous silicon layer is 8 to 12 cm.

[0027] Preferably, the first incubation temperature is room temperature, and the time is 2 to 3 hours; the second incubation temperature is room temperature, and the time is 0.5 to 1.5 hours.

[0028] The present invention provides a method for detecting SARS-CoV-2 antigen, comprising the following steps:

[0029] Covering the SPR sensor interface for detecting SARS-CoV-2 antigen with the sample to be tested and incubating;

[0030] The SPR angle of the SPR sensor interface obtained after the incubation is tested, and the content of the SARS-CoV-2 antigen in the test solution is obtained according to the SPR angle and a predetermined standard curve;

[0031] The standard curve is a linear relationship curve between the SPR angle and the SARS-CoV-2 antigen concentration.

[0032] The present invention provides an SPR sensor interface for detecting SARS-CoV-2 antigens, comprising a high refractive index glass, an indium tin oxide layer, an amorphous silicon layer, a gold layer and a Cu layer stacked in sequence. 3 (PO 4 ) 2 -BSA-GO nanoflower layer, the Cu 3 (PO 4 ) 2 -BSA-GO nanoflower layer includes Cu coated with ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflowers; the Cu 3 (PO 4 ) 2 -BSA-GO nanoflowers are obtained by precipitation reaction of copper sulfate, bovine serum albumin and graphene oxide in phosphate buffer; the refractive index of the high refractive index glass is 1.72-1.82; the Cu 3 (PO 4 ) 2 -BSA-GO nanoflower layer has a thickness of 20 to 40 nm. 3 (PO 4 ) 2 -BSA-GO nanoflowers fix ACE2 protein probes. The amide and carboxyl groups of the biological probes will coordinate with the metal ions of the nanoflowers. In addition, the biological probes also coordinate with Cu 2+ Combined, play an inductive role, further make Cu 3 (PO 4 ) 2 -BSA-GO nanoflowers are tightly "adhered" to the ACE2 protein probe; ACE2 protein can specifically bind to SARS-CoV-2 antigens. When the sample to be tested (such as aerosols, solutions) contains SARS-CoV-2 antigens, SARS-CoV-2 antigens bind to Cu 3 (PO 4 ) 2 -BSA-GO nanoflower layer surface, causing the SPR angle of the SPR sensor interface to change, thereby realizing the detection of SARS-CoV-2 antigen content in the sample to be tested. 3 (PO 4 )2 -BSA-GO nanoflowers have a microstructure that provides protection for ACE2 protein probes. They have specificity for bioactive molecules, good thermal stability, acid and alkali resistance, and are suitable as interface surface materials.

[0033] The SPR sensor interface for detecting SARS-CoV-2 antigen provided by the present invention has the following advantages: ① low detection limit, which can detect samples with a SARS-CoV-2 antigen concentration of 10nM; ② wide linear range for detection, which is 10nM to 10mM; ③ high stability, and its performance hardly changes within 30 days at room temperature; ④ little affected by temperature, with a low temperature response in the temperature range of 25 to 45°C, which can reduce the interference of temperature fluctuations in applications; ⑤ capable of regeneration and reuse. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the structure of the SPR sensor interface for detecting SARS-CoV-2 antigen;

[0035] Figure 2 Cu 3 (PO 4 ) 2 -SEM image of BSA-GO nanoflowers;

[0036] Figure 3 The relationship between the sensor detection antigen concentration and the SPR angle at the SPR sensor interface;

[0037] Figure 4 The detection results of the same standard sample on the surface of the SPR sensor at different temperatures;

[0038] Figure 5 It is the detection result of the SPR sensor surface for the same standard sample at different times;

[0039] Figure 6 For different Cu 3 (PO 4 ) 2 -Detection results of the SPR sensor interface for the same standard sample at different BSA-GO layer thicknesses. DETAILED DESCRIPTION

[0040] The present invention provides an SPR sensor interface for detecting SARS-CoV-2 antigens, comprising a high refractive index glass, an indium tin oxide layer, an amorphous silicon layer, a gold layer and a Cu layer stacked in sequence. 3 (PO 4 ) 2 -BSA-GO nanoflower layer, the Cu3 (PO 4 ) 2 -BSA-GO nanoflower layer includes Cu coated with ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflowers;

[0041] The Cu 3 (PO 4 ) 2 -BSA-GO nanoflowers were obtained by precipitation reaction of copper sulfate, bovine serum albumin and graphene oxide in phosphate buffer;

[0042] The refractive index of the high refractive index glass is 1.72 to 1.82, more preferably 1.75;

[0043] The Cu 3 (PO 4 ) 2 -The thickness of the BSA-GO nanoflower layer is 20 to 40 nm.

[0044] The SPR sensor interface for detecting SARS-CoV-2 antigen provided by the present invention includes high refractive index glass. In the present invention, the refractive index of the high refractive index glass is 1.72-1.82, more preferably 1.75; the thickness of the high refractive index glass is preferably 0.75-1 mm, more preferably 0.88 mm. In the present invention, the function of the high refractive index glass is to form an optical medium layer, which forms an evanescent wave under total reflection conditions, and is one of the production bases of the surface plasmon resonance effect, while reducing light loss and facilitating subsequent measurements.

[0045] The SPR sensor interface for detecting SARS-CoV-2 antigen provided by the present invention includes an indium tin oxide layer located on the surface of the high-refractive glass. In the present invention, the thickness of the indium tin oxide layer is preferably 60 to 100 nm, more preferably 80 to 90 nm. In the present invention, the function of the indium tin oxide layer is to form a transparent electrode layer. Indium tin oxide has excellent conductivity, thermal stability and graphic processing characteristics in addition to transparency.

[0046] The SPR sensor interface for detecting SARS-CoV-2 antigen provided by the present invention includes an amorphous silicon layer located on the surface of the indium tin oxide layer. In the present invention, the thickness of the amorphous silicon layer is preferably 80 to 120 nm, more preferably 100 nm. In the present invention, the function of the amorphous silicon layer is to utilize its lattice vibration to generate phonons to help photons excite electron movement, which is also one of the basic conditions for the generation of the surface plasmon resonance effect.

[0047] The SPR sensor interface for detecting SARS-CoV-2 antigen provided by the present invention includes a gold layer located on the surface of the amorphous silicon layer. In the present invention, the thickness of the gold layer is preferably 30 to 50 nm, more preferably 40 nm. In the present invention, the role of the gold layer is to enhance electronic oscillations and make the generated plasma wave more stable.

[0048] The SPR sensor interface for detecting SARS-CoV-2 antigen provided by the present invention comprises a Cu 3 (PO 4 ) 2 -BSA-GO nanoflower layer, the Cu 3 (PO 4 ) 2 -BSA-GO nanoflower layer includes Cu coated with ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflower. In the present invention, the ACE2 protein and Cu 3 (PO 4 ) 2 The mass ratio of -BSA-GO nanoflowers is preferably 1:6-10, more preferably 1:8.

[0049] In the present invention, the Cu 3 (PO 4 ) 2 -BSA-GO nanoflowers are obtained by precipitation reaction of copper sulfate, bovine serum albumin and graphene oxide in phosphate buffer. In the present invention, the mass ratio of copper sulfate, bovine serum albumin and graphene oxide is preferably 16:25-35:12.5-17.5, more preferably 16:30:15.

[0050] In the present invention, the Cu 3 (PO 4 ) 2 -The method for preparing BSA-GO nanoflowers preferably comprises the following steps:

[0051] The copper sulfate solution, phosphate buffer containing bovine serum albumin and graphene oxide were mixed to carry out precipitation reaction to obtain Cu 3 (PO 4 ) 2 -BSA-GO nanoflowers.

[0052] In the present invention, the concentration of the copper sulfate solution is preferably 100 mM; the concentration of the bovine serum albumin in the phosphate buffer containing bovine serum albumin and graphene oxide is preferably 1 mg / mL; the concentration of the graphene oxide is preferably 0.5 mg / mL; the concentration of the phosphate buffer is preferably 0.1 M, and the pH value is preferably 7.2. In the present invention, the mass ratio of the copper sulfate solution to the phosphate buffer containing bovine serum albumin and graphene oxide is preferably 1:25-35, more preferably 1:30.

[0053] The present invention preferably performs the precipitation reaction under static conditions. In the present invention, the precipitation reaction is preferably performed in a sterile constant temperature environment of 20 to 37°C, more preferably 25°C, and the precipitation reaction time is preferably 20 to 30 hours, more preferably 24 hours. In the present invention, the Cu 3 (PO 4 ) 2 The particle size of the -BSA-GO nanoflowers is preferably 4 to 8 μm, more preferably 6 μm.

[0054] Get the Cu 3 (PO 4 ) 2 -BSA-GO nanoflowers, the present invention preferably centrifuges and washes the obtained precipitation reaction solution. In the present invention, the centrifugal speed is preferably 8000-12000rpm, more preferably 10000rpm; the time is preferably 8-12min, more preferably 10min. In the present invention, the washing is preferably ultrapure water washing.

[0055] In the present invention, the Cu 3 (PO 4 ) 2 -The thickness of the BSA-GO nanoflower layer is 20 to 40 nm, more preferably 30 nm.

[0056] In the present invention, the indium tin oxide layer and the gold layer of the SPR sensor interface for detecting SARS-CoV-2 antigens are connected by conductive silver paint.

[0057] In the present invention, the structural schematic diagram of the SPR sensor interface for detecting SARS-CoV-2 antigen is as follows Figure 1 shown.

[0058] The present invention provides a method for preparing the above-mentioned SPR sensor interface for detecting SARS-CoV-2 antigens, comprising the following steps:

[0059] First magnetron sputtering indium tin oxide onto the surface of the high refractive index glass to form an indium tin oxide layer;

[0060] performing a second magnetron sputtering of amorphous silicon onto the surface of the indium tin oxide layer to form an amorphous silicon layer;

[0061] magnetron sputtering gold onto the surface of the amorphous silicon layer to form a gold layer;

[0062] ACE2 protein, Cu 3 (PO 4 ) 2 -BSA-GO nanoflowers were mixed with a buffer solution and incubated for the first time to obtain Cu coated with ACE2 protein probes. 3 (PO 4 ) 2 -BSA-GO nanoflowers;

[0063] The Cu coated with ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflowers were mixed with bovine serum albumin and incubated for the second time to obtain closed Cu coated with ACE2 protein probes. 3 (PO 4 ) 2 -BSA-GO nanoflowers;

[0064] The blocked Cu coated with ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflowers are deposited on the surface of the gold layer to obtain an SPR sensor interface for detecting SARS-CoV-2 antigens.

[0065] The present invention first magnetron sputters indium tin oxide onto the surface of the high refractive index glass to form an indium tin oxide layer. In the present invention, the gas for the first magnetron sputtering is preferably helium, the sputtering pressure is preferably 1-1.4 Pa, more preferably 1.2 Pa; the sputtering power is preferably 120-180 W, more preferably 150 W; the sputtering time is preferably 3-5 min, more preferably 4 min; the sputtering vacuum is preferably 6×10 -4 Pa; the distance between the sputtering target source and the high refractive index glass is preferably 8 to 12 cm, more preferably 10 cm.

[0066] The present invention forms an amorphous silicon layer by second magnetron sputtering of amorphous silicon onto the surface of the indium tin oxide layer. In the present invention, the gas for the second magnetron sputtering is helium, and the sputtering pressure is preferably 2-3 Pa, more preferably 2.5 Pa; the sputtering power is preferably 150-250 W, more preferably 200 W; the sputtering time is preferably 0.8-1.2 h, more preferably 1 h; the sputtering vacuum is preferably 6×10 -4 Pa; the distance between the sputtering target source and the indium tin oxide layer is preferably 8 to 12 cm, more preferably 10 cm.

[0067] In the present invention, gold is sputtered onto the surface of the amorphous silicon layer by the third magnetron sputtering to form a gold layer. In the present invention, the gas for the third magnetron sputtering is preferably helium, the sputtering pressure is preferably 2.5-3.5 Pa, more preferably 2 Pa; the sputtering power is preferably 240-260 W, more preferably 250 W; the sputtering time is preferably 30-50 s, more preferably 40 s; the sputtering vacuum is preferably 6×10 -4 Pa; the distance between the sputtering target source and the amorphous silicon layer is preferably 8 to 12 cm, more preferably 10 cm.

[0068] The present invention combines ACE2 protein, Cu 3 (PO 4 ) 2 -BSA-GO nanoflowers were mixed with a buffer solution and incubated for the first time to obtain Cu coated with ACE2 protein probes. 3 (PO 4 ) 2 -BSA-GO nanoflowers. In the present invention, the buffer solution is preferably a PBS buffer solution, the concentration of the PBS buffer solution is preferably 0.1 M, and the pH value is preferably 7.2.

[0069] In the present invention, the ACE2 protein, Cu 3 (PO 4 ) 2 The mass ratio of -BSA-GO nanoflowers to buffer solution is preferably 1:6 to 10:9, more preferably 1:8:9.

[0070] In the present invention, the mixing method is preferably shaking. In the present invention, the temperature of the first incubation is preferably room temperature, and the time is preferably 2 to 3 hours, more preferably 2.5 hours.

[0071] The present invention is to coat the Cu with ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflowers were mixed with bovine serum albumin and incubated for the second time to obtain closed Cu coated with ACE2 protein probes. 3 (PO 4 ) 2 -BSA-GO nanoflower. In the present invention, the Cu coated with ACE2 protein probe 3 (PO 4 ) 2 The mass ratio of -BSA-GO nanoflowers to bovine serum albumin is preferably 1:1-3, more preferably 1:2.

[0072] In the present invention, the temperature of the second incubation is preferably room temperature, and the time is preferably 0.5 to 1.5 hours, more preferably 1 hour.

[0073] The present invention is to enclose the Cu coated with the ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflowers are deposited on the surface of the gold layer to obtain an SPR sensor interface for detecting SARS-CoV-2 antigens. In the present invention, the deposition method is preferably electrodeposition. In the present invention, the electrodeposition preferably uses cyclic voltammetry, the potential scanning interval is preferably -0.2 to 1.6 V, the scanning rate is preferably 100 mV / s, and the duration is preferably 6 to 12 min, more preferably 9 min.

[0074] The present invention provides a method for detecting the above-mentioned SARS-CoV-2 antigen, comprising the following steps:

[0075] Covering the SPR sensor interface for detecting SARS-CoV-2 antigen with the sample to be tested, incubating, testing the SPR angle of the SPR sensor interface obtained after incubation, and obtaining the content of SARS-CoV-2 antigen in the test solution according to the SPR angle and a predetermined standard curve;

[0076] The standard curve is a linear relationship curve between the SPR angle and the SARS-CoV-2 antigen concentration.

[0077] The present invention covers the SPR sensor interface for detecting SARS-CoV-2 antigen with the sample to be tested and incubates. In the present invention, the sample to be tested is preferably an aerosol or an aqueous solution. In the present invention, the incubation temperature is preferably room temperature, and the time is preferably 10 to 30 minutes, more preferably 20 minutes.

[0078] In the present invention, the method for obtaining the standard curve preferably comprises the following steps:

[0079] Provide SARS-CoV-2 antigen standard solutions with known gradient concentrations;

[0080] The SARS-CoV-2 antigen standard solution with a known gradient concentration is used as a sample to be tested, covered on the SPR sensor interface for detecting the SARS-CoV-2 antigen, incubated, and the SPR angle of the SPR sensor interface is tested to obtain the SPR angle corresponding to the SARS-CoV-2 antigen standard solution with a known gradient concentration, and a standard curve is drawn with the concentration of the SARS-CoV-2 antigen standard solution as the abscissa and the SPR angle as the ordinate.

[0081] As a specific embodiment of the present invention, the concentrations of the SARS-CoV-2 antigen standard solutions with known gradient concentrations are 0.1nM, 1nM, 10nM, 100nM, 1μM, 10μM, 100μM, 1mM, 5mM, and 10mM, respectively;

[0082] The standard curve is Y=0.0023X+55.05, R 2 =0.9946, the detection limit is 10nM, and the linear range is 10nM to 10mM.

[0083] In the present invention, the SPR sensor interface for detecting SARS-CoV-2 antigens can be regenerated and reused. In the present invention, the method for regenerating the SPR sensor interface for detecting SARS-CoV-2 antigens preferably comprises the following steps:

[0084] The SPR sensor interface after detection is eluted using a glycine solution. In the present invention, the concentration of the glycine solution is preferably 100 mM, and the pH value is preferably 2. In the present invention, the glycine solution regenerates the fixed ACE2 protein probe.

[0085] The SPR sensor interface for detecting SARS-CoV-2 antigen and the detection method thereof provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0086] Example 1

[0087] The structure of the SPR sensor interface for detecting SARS-CoV-2 antigen is shown in Table 1:

[0088] Table 1 Structure of the SPR sensor interface for detecting SARS-CoV-2 antigens

[0089]

[0090] A method for preparing an SPR sensor interface for detecting SARS-CoV-2 antigens comprises the following steps:

[0091] ① Using high refractive index glass as the substrate, indium tin oxide is evenly covered on the substrate by magnetron sputtering to form a transparent electrode layer. The parameters of magnetron sputtering include: local vacuum is pumped to 6×10 -4 Pa, the sputtering gas is pure helium, the sputtering pressure is 1.2 Pa, the sputtering power is 150 W, the distance between the sputtering target source and the substrate is 10 cm, and the sputtering time is 4 min;

[0092] ② Then, amorphous silicon is uniformly covered on the transparent electrode layer prepared in ① by magnetron sputtering to form a semiconductor layer. The parameters of magnetron sputtering include: local vacuum is pumped to 6×10-4 Pa, the sputtering gas is pure helium, the sputtering pressure is 2.5 Pa, the sputtering power is 200 W, the distance between the sputtering target source and the substrate is 10 cm, and the sputtering time is 1 h;

[0093] ③ Then, gold is evenly covered on the semiconductor layer prepared in ② by magnetron sputtering to form a gold layer. The parameters of magnetron sputtering include: local vacuum is pumped to 6×10 -4 Pa, the sputtering gas is pure helium, the sputtering pressure is 2.4 Pa, the sputtering power is 250 W, the distance between the sputtering target source and the substrate is 10 cm, and the sputtering time is 40 s;

[0094] ④ The copper sulfate solution (100 mM) was mixed with phosphate buffer (PBS solution, 0.1 M, pH = 7.2) containing bovine serum albumin (BSA, 1 mg / mL) and graphene oxide (GO, 0.5 mg / mL) at a mass ratio of 1:30, and allowed to stand at 25 ° C in a sterile constant temperature environment for 24 h to grow Cu 3 (PO 4 ) 2 -BSA-GO nanoflowers;

[0095] ⑤ Prepare the Cu-containing 3 (PO 4 ) 2 - The BSA-GO nanoflower solution was centrifuged at 10,000 rpm for 10 min, after which the supernatant was removed and washed three times with ultrapure water. After each wash, the same centrifugation and supernatant removal steps were used;

[0096] Cu 3 (PO 4 ) 2 -SEM image of BSA-GO nanoflowers Figure 2 shown.

[0097] ⑥ Angiotensin converting enzyme 2 protein (ACE2 protein) was used as a probe and the prepared Cu 3 (PO 4 ) 2 -BSA-GO nanoflowers were added to PBS solution (0.1M, pH = 7.2), and the mass ratio (ACE2 protein: Cu 3 (PO 4 ) 2 -BSA-GO nanoflowers: PBS solution) were added in a ratio of 1:8:9, and after slight shaking, they were placed in a rotating incubator at room temperature for 2 h;

[0098] ⑦ Add BSA to solution ⑥ at a mass ratio of 1:1 to 3:1 (BSA: solution ⑥), with the optimal ratio being 2:1. After slight shaking, continue to culture in a rotating incubator at room temperature for 1 hour.

[0099] After step ⑧ and step ⑦ were completed, the mixture was centrifuged at 10,000 rpm in a refrigerated centrifuge for 4 minutes, and then the supernatant was removed. The mixture was washed three times with a non-ionic amphoteric buffer (0.1 M, pH = 7.2). After each wash, the same steps were used for centrifugation and the supernatant was removed to obtain a Cu 2+-blocked ACE2 protein probe fixed thereon. 3 (PO 4 ) 2 -BSA-GO nanoflowers;

[0100] ⑨ The blocked Cu with ACE2 protein probe fixed on it 3 (PO 4 ) 2 -BSA-GO nanoflowers are naturally deposited on the gold layer according to the designed thickness;

[0101] ⑩Finally, the electrodes were connected to the transparent electrode layer and the gold layer using conductive silver paint to complete the preparation of the SPR sensor interface for SARS-CoV-2 antigen detection.

[0102] Example 2

[0103] All the following examples and verification experiments should cover the prepared SPR sensor interface with an inert buffer solution before being carried out. The inert buffer solution should be replaced with a solution containing the tested novel coronavirus SARS-CoV-2 antigen when in use, and the solution containing the tested novel coronavirus SARS-CoV-2 antigen should be replaced with the prepared SPR sensor interface for 10 to 30 minutes before each detection. If the prepared SPR sensor interface is reused, it should be cleaned with an inert buffer solution after emptying the tested solution each time, and the surface of the prepared SPR sensor interface should be eluted with a 100mM, pH=2 glycine solution to regenerate the fixed capture protein.

[0104] (1) Verification of linear range and detection limit:

[0105] Ten concentrations of SARS-CoV-2 antigen standard samples of 0.1nM, 1nM, 10nM, 100nM, 1μM, 10μM, 100μM, 1mM, 5mM, and 10mM were prepared and tested using sensors containing the designed SPR sensor interface. The results are shown in Figure 3 As shown, with the increase of the concentration of the antigen being tested, the SPR angle also increases accordingly, with a detection limit as low as 10 nM and a linear range of 10 nM to 10 mM.

[0106] (2) Verification of the impact of common operating temperatures on sensor interface performance

[0107] The prepared SPR sensor interface for detecting SARS-CoV-2 antigen in aerosol was used to detect the standard sample with a concentration of 1 μM of the novel coronavirus SARS-CoV-2 antigen at five temperatures: 25°C, 30°C, 35°C, 40°C, and 45°C. The results are as follows: Figure 4 As shown, at 25-45°C, the prepared SPR sensor interface is less affected by temperature, which can reduce the detection error caused by temperature fluctuations in applications.

[0108] (3) Verification of the interface stability of the prepared SPR sensor:

[0109] The prepared SPR sensor interface for detecting SARS-CoV-2 antigen in aerosol was stored as required, and the standard sample with a concentration of 1 μM of the same novel coronavirus SARS-CoV-2 antigen was tested on day 0, day 5, day 10, day 15, day 20, and day 30. The results were as follows: Figure 5 As shown, with the increase of time, the SPR angle of the same sample only decreases slightly, indicating its good stability and its performance hardly changes within 30 days at room temperature.

[0110] Example 3

[0111] Different surface material thicknesses correspond to different relationships between SPR angle and refractive index. Therefore, in order to verify the Cu 3 (PO 4 ) 2 -BSA-GO nanomaterials with the best thickness, prepared five kinds of Cu nanoparticles with thickness of 10nm, 20nm, 30nm, 40nm and 50nm respectively. 3 (PO 4 ) 2 -BSA-GO layer for SARS-CoV-2 antigen detection in aerosol SPR sensor interface, and using different thicknesses of Cu 3 (PO 4 ) 2 -BSA-GO layer sensor was used to test the standard sample with a concentration of 1 μM of the new coronavirus SARS-CoV-2 antigen. The results are as follows Figure 6 As shown, no matter what thickness Cu 3 (PO 4 ) 2 -BSA-GO layer surface, the refractive index reaches the lowest when the SPR angle is about 55°, but when the thickness is 30nm, the refractive index is almost 0 when the SPR angle is at the lowest point. As the thickness gradually increases from 30nm, the refractive index at this SPR angle also gradually increases, indicating that the selection of Cu with a thickness of 20nm to 40nm 3 (PO4 ) 2 -BSA-GO layer as the designed SPR sensor interface can achieve better detection effect, in which Cu 3 (PO 4 ) 2 -The optimal thickness of the BSA-GO layer is 30 nm.

[0112] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An SPR sensor interface for detecting SARS-CoV-2 antigens, comprising a high refractive index glass, an indium tin oxide layer, an amorphous silicon layer, a gold layer and a Cu layer stacked in sequence 3 (PO 4 ) 2 -BSA-GO nanoflower layer, the Cu 3 (PO 4 ) 2 -BSA-GO nanoflower layer includes Cu coated with ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflowers; The Cu 3 (PO 4 ) 2 -BSA-GO nanoflowers were obtained by precipitation reaction of copper sulfate, bovine serum albumin and graphene oxide in phosphate buffer; The refractive index of the high refractive index glass is 1.72-1.82; The Cu 3 (PO 4 ) 2 -The thickness of the BSA-GO nanoflower layer is 20~40 nm; The thickness of the high refractive index glass is 0.75-1 mm; The thickness of the indium tin oxide layer is 60-100 nm; The thickness of the amorphous silicon layer is 80-120 nm; The thickness of the gold layer is 30-50 nm.

2. The SPR sensor interface for detecting SARS-CoV-2 antigen according to claim 1, It is characterized in that The mass ratio of copper sulfate, bovine serum albumin and graphene oxide is 16:25-35:12.5-17.5; The temperature of the precipitation reaction is 20-37°C and the time is 20-30 h.

3. The SPR sensor interface for detecting SARS-CoV-2 antigen according to claim 1, It is characterized in that The ACE2 protein and Cu 3 (PO 4 ) 2 -The mass ratio of BSA-GO nanoflowers is 1:6~10.

4. The method for preparing the SPR sensor interface for detecting SARS-CoV-2 antigen according to any one of claims 1 to 3, The following steps are involved: First magnetron sputtering indium tin oxide onto the surface of the high refractive index glass to form an indium tin oxide layer; performing a second magnetron sputtering of amorphous silicon onto the surface of the indium tin oxide layer to form an amorphous silicon layer; magnetron sputtering gold onto the surface of the amorphous silicon layer to form a gold layer; ACE2 protein, Cu 3 (PO 4 ) 2 -BSA-GO nanoflowers were mixed with a buffer solution and incubated for the first time to obtain Cu coated with ACE2 protein probes. 3 (PO 4 ) 2 -BSA-GO nanoflowers; The Cu coated with ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflowers were mixed with bovine serum albumin and incubated for the second time to obtain closed Cu coated with ACE2 protein probes. 3 (PO 4 ) 2 -BSA-GO nanoflowers; The blocked Cu coated with ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflowers are deposited on the surface of the gold layer to obtain an SPR sensor interface for detecting SARS-CoV-2 antigens.

5. The preparation method according to claim 4, It is characterized in that The gas for the first magnetron sputtering is helium, the sputtering pressure is 1-1.4 Pa, the sputtering power is 120-180 W, the sputtering time is 3-5 min, and the sputtering vacuum is 6×10 -4 Pa; the distance between the sputtering target source and the high refractive index glass is 8~12 cm.

6. The preparation method according to claim 4, It is characterized in that The gas for the second magnetron sputtering is helium, the sputtering pressure is 2-3 Pa, the sputtering power is 150-250 W, the sputtering time is 0.8-1.2 h, and the sputtering vacuum is 6×10 -4 Pa; the distance between the sputtering target source and the indium tin oxide layer is 8~12 cm.

7. The preparation method according to claim 4, It is characterized in that The gas for the third magnetron sputtering is helium, the sputtering pressure is 2.5-3.5 Pa, the sputtering power is 240-260 W, the sputtering time is 30-50 s, and the sputtering vacuum is 6×10 -4 Pa; the distance between the sputtering target source and the amorphous silicon layer is 8~12 cm.

8. The preparation method according to claim 4, It is characterized in that The first incubation temperature is room temperature, and the time is 2-3 h; the second incubation temperature is room temperature, and the time is 0.5-1.5 h.

9. A method for detecting SARS-CoV-2 antigens for non-diagnostic purposes, The following steps are involved: Covering the SPR sensor interface for detecting SARS-CoV-2 antigens according to any one of claims 1 to 3 or the SPR sensor interface for detecting SARS-CoV-2 antigens prepared by the preparation method according to any one of claims 4 to 8 with the sample to be tested, and incubating; the sample to be tested is an aerosol or an aqueous solution; The SPR angle of the SPR sensor interface obtained after the incubation is tested, and the content of the SARS-CoV-2 antigen in the test solution is obtained according to the SPR angle and a predetermined standard curve; The standard curve is a linear relationship curve between the SPR angle and the SARS-CoV-2 antigen concentration.

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