CN116106546A - SPR sensor interface for detecting SARS-CoV-2 antigen and its detecting method - Google Patents

SPR sensor interface for detecting SARS-CoV-2 antigen and its detecting method Download PDF

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CN116106546A
CN116106546A CN202310133712.XA CN202310133712A CN116106546A CN 116106546 A CN116106546 A CN 116106546A CN 202310133712 A CN202310133712 A CN 202310133712A CN 116106546 A CN116106546 A CN 116106546A
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陈真诚
龚柳宇
赵飞骏
沈亚
曾茂强
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Guilin University of Electronic Technology
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Abstract

The 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. The invention uses Cu 3 (PO 4 ) 2 The BSA-GO nanoflower fixes an ACE2 protein probe, the amido and carboxyl of the ACE2 protein probe can coordinate with copper metal ions of the nanoflower, and in addition, the biological probe also coordinates with Cu 2+ Bind to exert an induction effect, and further cause Cu to be present 3 (PO 4 ) 2 -BSA-GO nanoflower is tightly "tethered" to ACE2 protein probe; ACE2 protein can specifically bind to SARS-CoV-2 antigen and bind SARS-CoV-2 antigen to Cu 3 (PO 4 ) 2 ‑BThe surface of the SA-GO nano flower layer causes the SPR angle of the SPR sensor interface to change, thereby realizing the detection of the SARS-CoV-2 antigen content in the sample to be detected.

Description

SPR sensor interface for detecting SARS-CoV-2 antigen and its detecting method
Technical Field
The invention relates to the technical field of SPR sensors, in particular to an SPR sensor interface for detecting SARS-CoV-2 antigen and a detection method thereof.
Background
The two currently used methods for detecting the novel coronaviruses are molecular diagnosis techniques based on Polymerase Chain Reaction (PCR), nucleic acid hybridization and second generation sequencing techniques and immunodiagnosis techniques for detecting antigens or antibodies generated in patients after exposure to the novel coronaviruses, respectively. The former output results are long in time consumption, and the latter are low in accuracy, and cannot be matched with the requirements of detection of new coronaviruses in aerosols in air in a large space. Therefore, a detection method which is quick in real time, good in stability and high in accuracy and can process a large number of accumulated samples needs to be found.
The Surface Plasmon Resonance (SPR) detection technology is a non-labeling detection method, is used for detecting between real-time, high-sensitivity and measurable biological samples, and well meets the requirements of detecting new coronaviruses in aerosol in public space environment. However, the interface of the conventional SPR sensor is usually used for fixing the designed biological probe in a chemical combination manner through Au-S bond, and the fixing manner is easy to fall off.
Disclosure of Invention
In view of the above, the present invention aims to provide an SPR sensor interface for detecting SARS-CoV-2 antigen and a detection method thereof. The SPR sensor interface provided by the invention can effectively fix biological probes and realize accurate detection of SARS-CoV-2 antigen.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an SPR sensor interface for detecting SARS-CoV-2 antigen, which comprises high refractive index glass, indium tin oxide layer, amorphous silicon layer, gold layer and Cu laminated in turn 3 (PO 4 ) 2 -BSA-GO nanoflower layer, cu 3 (PO 4 ) 2 the-BSA-GO nanoflower layer comprises Cu coated with ACE2 protein probes 3 (PO 4 ) 2 -BSA-GO nanoflower;
the Cu is 3 (PO 4 ) 2 The BSA-GO nanoflower is obtained by precipitation reaction of copper sulfate, bovine serum albumin and graphene oxide in phosphate buffer solution;
the refractive index of the high refractive index glass is 1.72-1.82;
the Cu is 3 (PO 4 ) 2 The thickness of the BSA-GO nano-flower layer is 20-40 nm.
Preferably, 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.
Preferably, the mass ratio of the copper sulfate to the bovine serum albumin to the graphene oxide is 16:25-35:12.5-17.5;
the temperature of the precipitation reaction is 20-37 ℃ and the time is 20-30 h.
Preferably, the ACE2 protein and Cu 3 (PO 4 ) 2 The mass ratio of the BSA-GO nanoflowers is 1:6-10.
The invention provides a preparation method of an SPR sensor interface for detecting SARS-CoV-2 antigen, which comprises the following steps:
sputtering indium tin oxide onto the surface of high refractive index glass by a first magnetron sputtering method to form an indium tin oxide layer;
performing second magnetron sputtering on amorphous silicon to the surface of the indium tin oxide layer to form an amorphous silicon layer;
sputtering gold on the surface of the amorphous silicon layer by a third magnetron sputtering method to form a gold layer;
ACE2 protein, cu 3 (PO 4 ) 2 Mixing the BSA-GO nanoflower with a buffer solution, and performing first incubation to obtain Cu coated with ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflower;
cu coated with ACE2 protein probe 3 (PO 4 ) 2 Mixing BSA-GO nanoflower with bovine serum albumin, and performing second incubation to obtain blockedCu coated with ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflower;
cu coating the closed Cu coated with ACE2 protein probe 3 (PO 4 ) 2 And depositing BSA-GO nanoflower on the surface of the gold layer to obtain an SPR sensor interface for detecting SARS-CoV-2 antigen.
Preferably, the gas of the first magnetron sputtering is helium, the sputtering pressure is 1-1.4 Pa, the sputtering power is 120-180W, the sputtering time is 3-5 min, and the sputtering vacuum degree is 6 multiplied by 10 -4 Pa; the distance between the sputtering target source and the high refractive index glass is 8-12 cm.
Preferably, the gas of the second magnetron sputtering is helium, the sputtering pressure is 2-3 Pa, the sputtering power is 150-250W, the sputtering time is 0.8-1.2 h, and the sputtering vacuum degree is 6 multiplied by 10 -4 Pa; the distance between the sputtering target source and the indium tin oxide layer is 8-12 cm.
Preferably, the gas of the third magnetron sputtering is helium, the sputtering pressure is 2.5-3.5 Pa, the sputtering power is 240-260W, the sputtering time is 30-50 s, and the sputtering vacuum degree is 6 multiplied by 10 -4 Pa; the distance between the sputtering target source and the amorphous silicon layer is 8-12 cm.
Preferably, the temperature of the first incubation is room temperature and the time is 2-3 hours; the temperature of the second incubation is room temperature and the time is 0.5-1.5 h.
The invention provides a detection method of SARS-CoV-2 antigen, comprising the following steps:
covering the SPR sensor interface for detecting SARS-CoV-2 antigen with the sample to be detected, and incubating;
testing the SPR angle of the SPR sensor interface obtained after incubation, and obtaining the content of SARS-CoV-2 antigen in the solution to be tested according to the SPR angle and a preset standard curve;
the standard curve is a linear relationship curve of SPR angle and SARS-CoV-2 antigen concentration.
The invention provides an SPR sensor interface for detecting SARS-CoV-2 antigen, which comprises high refractive index glass, indium tin oxide layer, amorphous silicon layer, gold layer and Cu laminated in turn 3 (PO 4 ) 2 -BSA-GO nanoflower layer, cu 3 (PO 4 ) 2 the-BSA-GO nanoflower layer comprises Cu coated with ACE2 protein probes 3 (PO 4 ) 2 -BSA-GO nanoflower; the Cu is 3 (PO 4 ) 2 The BSA-GO nanoflower is obtained by precipitation reaction of copper sulfate, bovine serum albumin and graphene oxide in phosphate buffer solution; the refractive index of the high refractive index glass is 1.72-1.82; the Cu is 3 (PO 4 ) 2 The thickness of the BSA-GO nano-flower layer is 20-40 nm. The invention uses Cu 3 (PO 4 ) 2 The BSA-GO nanoflower immobilized ACE2 protein probe has the amino, carboxyl and other groups coordinated with the metal ion of the nanoflower, and the biological probe is also coordinated with Cu 2+ Bind to exert an induction effect, and further cause Cu to be present 3 (PO 4 ) 2 -BSA-GO nanoflower is tightly "tethered" to ACE2 protein probe; ACE2 protein can be specifically combined with SARS-CoV-2 antigen, when the sample (such as aerosol and solution) to be tested contains SARS-CoV-2 antigen, SARS-CoV-2 antigen is combined with Cu 3 (PO 4 ) 2 The surface of the BSA-GO nano-flower layer causes the SPR angle of the SPR sensor interface to change, thereby realizing the detection of the SARS-CoV-2 antigen content in the sample to be detected. At the same time, cu 3 (PO 4 ) 2 The BSA-GO nanoflower has a microstructure with a protective function provided by an ACE2 protein probe, has specificity to bioactive molecules, has good heat stability and acid and alkali resistance, and is suitable for being used as an interface surface layer material.
The SPR sensor interface for detecting SARS-CoV-2 antigen provided by the invention has the following advantages: (1) a sample having a low Detection limit (Detection limit) and a SARS-CoV-2 antigen concentration of 10nM can be detected; (2) the range of Linearity for detection is 10nM to 10mM; (3) the stability is high, and the performance of the composition is hardly changed within 30 days at room temperature; (4) the temperature influence is small, and the temperature response is low in the temperature range of 25-45 ℃, so that the interference of temperature fluctuation in application can be reduced; (5) can be regenerated and reused.
Drawings
FIG. 1 is a schematic structural diagram of SPR sensor interface for detecting SARS-CoV-2 antigen;
FIG. 2 is Cu 3 (PO 4 ) 2 SEM images of BSA-GO nanoflowers;
FIG. 3 is a graph showing the relationship between the concentration of the detected antigen by the sensor at the interface of the SPR sensor and the SPR angle;
FIG. 4 shows the detection results of SPR sensor surfaces on the same standard sample at different temperatures;
FIG. 5 shows the detection results of SPR sensor surfaces on the same standard sample at different times;
FIG. 6 shows different Cu 3 (PO 4 ) 2 Detection results of SPR sensor interface on the same standard sample under BSA-GO layer thickness.
Detailed Description
The invention provides an SPR sensor interface for detecting SARS-CoV-2 antigen, which comprises high refractive index glass, indium tin oxide layer, amorphous silicon layer, gold layer and Cu laminated in turn 3 (PO 4 ) 2 -BSA-GO nanoflower layer, cu 3 (PO 4 ) 2 the-BSA-GO nanoflower layer comprises Cu coated with ACE2 protein probes 3 (PO 4 ) 2 -BSA-GO nanoflower;
the Cu is 3 (PO 4 ) 2 The BSA-GO nanoflower is obtained by precipitation reaction of copper sulfate, bovine serum albumin and graphene oxide in phosphate buffer solution;
the refractive index of the high refractive index glass is 1.72 to 1.82, more preferably 1.75;
the Cu is 3 (PO 4 ) 2 The thickness of the BSA-GO nano-flower layer is 20-40 nm.
The SPR sensor interface for detecting SARS-CoV-2 antigen includes high refractive index glass. In the present invention, the refractive index of the high refractive index glass is 1.72 to 1.82, more preferably 1.75; the thickness of the high refractive index glass is preferably 0.75 to 1mm, more preferably 0.88mm. In the invention, the high refractive index glass has the function of forming an optical medium layer, forming an evanescent wave under the condition of total reflection, being one of the production bases of the surface plasmon resonance effect, reducing the light loss and facilitating the subsequent measurement.
The SPR sensor interface for detecting SARS-CoV-2 antigen provided by the invention comprises an indium tin oxide layer positioned on the surface of the high refractive glass. In the present invention, the thickness of the indium tin oxide layer is preferably 60 to 100nm, more preferably 80 to 90nm. In the present invention, the indium tin oxide layer functions to form a transparent electrode layer, and indium tin oxide has excellent conductivity, thermal stability and patterning characteristics in addition to transparency.
The SPR sensor interface for detecting SARS-CoV-2 antigen includes an amorphous silicon layer on the surface of the indium tin oxide layer. In the present invention, the thickness of the amorphous silicon layer is preferably 80 to 120nm, more preferably 100nm. In the invention, the amorphous silicon layer is used for generating phonons to help photons to excite electron motion by using lattice vibration, which is also one of the basic conditions for generating surface plasmon resonance effect.
The SPR sensor interface for detecting SARS-CoV-2 antigen includes gold layer on the surface of the amorphous silicon layer. In the present invention, the thickness of the gold layer is preferably 30 to 50nm, more preferably 40nm. In the invention, the gold layer has the function of enhancing electron oscillation and stabilizing the generated plasma wave.
The SPR sensor interface for detecting SARS-CoV-2 antigen includes Cu on the surface of the gold layer 3 (PO 4 ) 2 -BSA-GO nanoflower layer, cu 3 (PO 4 ) 2 the-BSA-GO nanoflower layer comprises Cu coated with ACE2 protein probes 3 (PO 4 ) 2 -BSA-GO nanoflower. In the present invention, the ACE2 protein and Cu 3 (PO 4 ) 2 The mass ratio of the BSA-GO nanoflowers is preferably 1:6-10, more preferably 1:8.
In the present invention, the Cu 3 (PO 4 ) 2 The BSA-GO nanoflower is obtained by precipitation reaction of copper sulfate, bovine serum albumin and graphene oxide in phosphate buffer. In the invention, the mass ratio of the copper sulfate to the bovine serum albumin to the graphene oxide is preferably 16:25-35:12.5-17.5, more preferably 16:30:15。
In the present invention, the Cu 3 (PO 4 ) 2 -BSA-GO nanoflower preparation method, preferably comprising the steps of:
mixing a copper sulfate solution, a phosphate buffer solution containing bovine serum albumin and graphene oxide, and performing precipitation reaction to obtain Cu 3 (PO 4 ) 2 -BSA-GO nanoflower.
In the present invention, the concentration of the copper sulfate solution is preferably 100mM; the concentration of the bovine serum albumin in the phosphate buffer solution containing the bovine serum albumin and the graphene oxide is preferably 1mg/mL; the concentration of the graphene oxide is preferably 0.5mg/mL; the concentration of the phosphate buffer is preferably 0.1M and the pH is preferably 7.2. In the present invention, the mass ratio of the copper sulfate solution to the phosphate buffer solution containing bovine serum albumin and graphene oxide is preferably 1:25 to 35, more preferably 1:30.
The precipitation reaction is preferably carried out under standing conditions. In the present invention, the precipitation reaction is preferably performed under an aseptic constant temperature environment of 20 to 37 ℃, more preferably 25 ℃, and the time of the precipitation reaction 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. Mu.m, more preferably 6. Mu.m.
Obtaining the Cu 3 (PO 4 ) 2 After the BSA-GO nanoflower, the obtained precipitation reaction solution is preferably centrifuged and washed. In the present invention, the rate of centrifugation is preferably 8000 to 12000rpm, more preferably 10000rpm; the time is preferably 8 to 12 minutes, more preferably 10 minutes. In the present invention, the washing is preferably ultrapure water washing.
In the present invention, the Cu 3 (PO 4 ) 2 The thickness of the BSA-GO nanoflower layer is 20-40 nm, more preferably 30nm.
In the invention, the indium tin oxide layer and the gold layer of the SPR sensor interface for detecting SARS-CoV-2 antigen are connected through conductive silver paint.
In the present invention, the structure of the SPR sensor interface for detecting SARS-CoV-2 antigen is schematically shown in FIG. 1.
The invention provides a preparation method of an SPR sensor interface for detecting SARS-CoV-2 antigen, which comprises the following steps:
sputtering indium tin oxide onto the surface of high refractive index glass by a first magnetron sputtering method to form an indium tin oxide layer;
performing second magnetron sputtering on amorphous silicon to the surface of the indium tin oxide layer to form an amorphous silicon layer;
sputtering gold on the surface of the amorphous silicon layer by a third magnetron sputtering method to form a gold layer;
ACE2 protein, cu 3 (PO 4 ) 2 Mixing the BSA-GO nanoflower with a buffer solution, and performing first incubation to obtain Cu coated with ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflower;
cu coated with ACE2 protein probe 3 (PO 4 ) 2 Mixing the BSA-GO nanoflower with bovine serum albumin, and performing second incubation to obtain the closed Cu coated with ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflower;
cu coating the closed Cu coated with ACE2 protein probe 3 (PO 4 ) 2 And depositing BSA-GO nanoflower on the surface of the gold layer to obtain an SPR sensor interface for detecting SARS-CoV-2 antigen.
The invention forms an indium tin oxide layer by sputtering indium tin oxide onto the surface of high refractive index glass by a first magnetron sputtering method. In the present invention, the gas of the first magnetron sputtering is preferably helium, and the sputtering pressure is preferably 1 to 1.4Pa, more preferably 1.2Pa; the sputtering power is preferably 120 to 180W, more preferably 150W; the sputtering time is preferably 3 to 5 minutes, more preferably 4 minutes; the sputtering vacuum degree is preferably 6×10 -4 Pa; the distance between the sputtering target source and the high refractive index glass is preferably 8 to 12cm, more preferably 10cm.
The amorphous silicon is subjected to second magnetron sputtering to the surface of the indium tin oxide layer to form the amorphous silicon layer. In the invention, the gas of the second magnetron sputtering is helium, and the sputtering pressure is preferably 2-3 Pa, more preferably 2.5Pa; the sputtering power is preferably 150 to 250WMore preferably 200W; the sputtering time is preferably 0.8 to 1.2 hours, more preferably 1 hour; the sputtering vacuum degree is preferably 6×10 -4 Pa; the distance between the sputtering target source and the indium tin oxide layer is preferably 8 to 12cm, more preferably 10cm.
The invention sputters gold third magnetron onto the surface of the amorphous silicon layer to form a gold layer. In the present invention, the gas of the third magnetron sputtering is preferably helium, and the sputtering pressure is preferably 2.5 to 3.5Pa, more preferably 2Pa; the sputtering power is preferably 240 to 260W, more preferably 250W; the sputtering time is preferably 30 to 50s, more preferably 40s; the sputtering vacuum degree is preferably 6×10 -4 Pa; the distance between the sputtering target source and the amorphous silicon layer is preferably 8 to 12cm, more preferably 10cm.
The invention uses ACE2 protein and Cu 3 (PO 4 ) 2 Mixing the BSA-GO nanoflower with a buffer solution, and performing first incubation to obtain Cu coated with ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflower. In the present invention, the buffer solution is preferably a PBS buffer solution, the concentration of the PBS buffer solution is preferably 0.1M, and the pH value is preferably 7.2.
In the present invention, the ACE2 protein, cu 3 (PO 4 ) 2 The mass ratio of the BSA-GO nanoflower to the buffer solution is preferably 1:6-10:9, more preferably 1:8:9.
In the present invention, the mixing means is preferably oscillation. 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.
The invention coats the Cu coated with the ACE2 protein probe 3 (PO 4 ) 2 Mixing the BSA-GO nanoflower with bovine serum albumin, and performing second incubation to obtain the closed Cu coated with ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflower. In the invention, the Cu coated with ACE2 protein probe 3 (PO 4 ) 2 The mass ratio of the BSA-GO nanoflower to the bovine serum albumin is preferably 1:1 to 3, more preferably 1:2.
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.
The invention coats the Cu with ACE2 protein probe 3 (PO 4 ) 2 And depositing BSA-GO nanoflower on the surface of the gold layer to obtain an SPR sensor interface for detecting SARS-CoV-2 antigen. In the present invention, the deposition means is preferably electrodeposition. In the present invention, the electrodeposition is preferably performed by cyclic voltammetry, the potential sweep interval is preferably-0.2 to 1.6V, the sweep rate is preferably 100mV/s, and the duration is preferably 6 to 12min, more preferably 9min.
The invention provides a method for detecting SARS-CoV-2 antigen, which comprises the following steps:
covering a sample to be detected on the SPR sensor interface for detecting SARS-CoV-2 antigen, incubating, testing the SPR angle of the SPR sensor interface obtained after incubation, and obtaining the content of SARS-CoV-2 antigen in the solution to be detected according to the SPR angle and a preset standard curve;
the standard curve is a linear relationship curve of SPR angle and SARS-CoV-2 antigen concentration.
The invention covers the SPR sensor interface for detecting SARS-CoV-2 antigen with the sample to be detected, and incubates. In the present invention, the sample to be measured is preferably an aerosol or an aqueous solution. In the present invention, the temperature of the incubation is preferably room temperature, and the time is preferably 10 to 30min, more preferably 20min.
In the present invention, the method for obtaining a standard curve preferably includes the steps of:
providing a standard solution of SARS-CoV-2 antigen at a known concentration gradient;
the standard solution of SARS-CoV-2 antigen with known gradient concentration is used as a sample to be tested, covered on an SPR sensor interface for detecting SARS-CoV-2 antigen, incubation is carried out, the SPR angle of the SPR sensor interface is tested, the SPR angle corresponding to the standard solution of SARS-CoV-2 antigen with known gradient concentration is obtained, the concentration of the standard solution of SARS-CoV-2 antigen is used as an abscissa, and the SPR angle is used as an ordinate, and a standard curve is drawn.
As a specific example of the present invention, the concentration of the standard solution of SARS-CoV-2 antigen of the gradient known concentration is 0.1nM, 1nM, 10nM, 100nM, 1. Mu.M, 10. Mu.M, 100. Mu.M, 1mM, 5mM, 10mM, respectively;
the standard curve is y=0.0023x+55.05, r 2 = 0.9946, detection limit is 10nM, linear range is 10nM to 10mM.
In the present invention, the SPR sensor interface for detecting SARS-CoV-2 antigen can be regenerated and reused. In the present invention, the method for regenerating the SPR sensor interface for detecting SARS-CoV-2 antigen preferably comprises the following steps:
eluting the detected SPR sensor interface by using glycine solution. In the present invention, the concentration of the glycine solution is preferably 100mM and the pH value is preferably 2. In the present invention, the glycine solution regenerates the immobilized ACE2 protein probe.
The SPR sensor interface for detecting SARS-CoV-2 antigen and the detection method thereof according to the present invention will be described in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The structure of the SPR sensor interface for detecting SARS-CoV-2 antigen is shown in Table 1:
TABLE 1 Structure of SPR sensor interface for detecting SARS-CoV-2 antigen
Figure SMS_1
The preparation method of the SPR sensor interface for detecting SARS-CoV-2 antigen comprises the following steps:
(1) using high refractive index glass as a substrate, uniformly covering indium tin oxide on the substrate by magnetron sputtering to form a transparent electrode layer, wherein the parameters of the magnetron sputtering comprise: vacuum-pumping to 6×10 -4 Pa, the sputtering gas is pure helium, the sputtering air pressure is 1.2Pa, the sputtering power is 150W, the distance between a sputtering target source and a substrate is 10cm, and the sputtering time is 4min;
(2) and then uniformly covering amorphous silicon on the transparent electrode layer prepared in the step (1) by magnetron sputtering to form a semiconductor layer, wherein the parameters of the magnetron sputtering comprise: local vacuumPumping to 6X 10 -4 Pa, the sputtering gas is pure helium, the sputtering air pressure is 2.5Pa, the sputtering power is 200W, the distance between a sputtering target source and a substrate is 10cm, and the sputtering time is 1h;
(3) and then uniformly covering the gold on the semiconductor layer prepared in the step (2) by magnetron sputtering to form a gold layer, wherein the parameters of the magnetron sputtering comprise: vacuum-pumping to 6×10 -4 Pa, the sputtering gas is pure helium, the sputtering air pressure is 2.4Pa, the sputtering power is 250W, the distance between a sputtering target source and a substrate is 10cm, and the sputtering time is 40s;
(4) copper sulfate solution (100 mM) was mixed with phosphate buffer (PBS solution, 0.1m, ph=7.2) containing bovine serum albumin (BSA, 1 mg/mL) and graphene oxide (GO, 0.5 mg/mL) in a mass ratio of 1:30, and standing for 24h at 25deg.C under aseptic constant temperature to grow Cu 3 (PO 4 ) 2 -BSA-GO nanoflower;
(5) cu-containing material prepared in the step (4) 3 (PO 4 ) 2 The solution of BSA-GO nanoflower was centrifuged at 10000rpm for 10min, after which the supernatant was removed and washed 3 times with ultrapure water, after each washing the same procedure was used for centrifugation and supernatant removal;
the Cu obtained 3 (PO 4 ) 2 SEM images of BSA-GO nanoflowers are shown in figure 2.
(6) Using angiotensin converting enzyme 2 protein (ACE 2 protein) as probe and prepared Cu 3 (PO 4 ) 2 Adding BSA-GO nanoflower to PBS solution (0.1M, pH=7.2) in a mass ratio (ACE 2 protein: cu 3 (PO 4 ) 2 -BSA-GO nanoflower: PBS solution) of 1:8:9, adding, slightly shaking, and then placing the mixture in a rotary incubator at room temperature for 2 hours;
(7) then the mass ratio (BSA (6) solution) is 1:1 to 3:1, most preferably 2:1 adding BSA into the solution (6), slightly oscillating, and then continuously placing the mixture into a rotary incubator to culture for 1h at room temperature;
(8) centrifuging in a refrigerated centrifuge at 10000rpm for 4min after the completion of step (7), removing supernatant, washing 3 times with nonionic amphoteric buffer (0.1M, pH=7.2), and using the same steps after each washingCentrifuging and removing supernatant to obtain Cu with immobilized ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflower;
(9) cu with sealed ACE2 protein probe immobilized thereon 3 (PO 4 ) 2 The BSA-GO nanoflower is naturally deposited on the gold layer according to the designed thickness;
and finally, connecting the electrode to the transparent electrode layer and the gold layer by using conductive silver paint to finish the preparation of an SPR sensor interface for SARS-CoV-2 antigen detection.
Example 2
All the following examples and verification experiments are carried out by covering the prepared SPR sensor interface with an inert buffer solution before the experiment is carried out, replacing the inert buffer solution with a solution containing the SARS-CoV-2 antigen of the novel coronavirus to be detected, and incubating the replacement of the solution containing the SARS-CoV-2 antigen of the novel coronavirus to be detected with the prepared SPR sensor interface for 10-30 min before each detection. The SPR sensor interface prepared was reused, washed with inert buffer after each use with emptying of the solution to be tested, and eluted with 100mm glycine solution at ph=2 to regenerate immobilized capture protein.
(1) Verification of the linear range and detection limit:
SARS-CoV-2 antigen standard samples were prepared at ten concentrations of 0.1nM, 1nM, 10nM, 100nM, 1. Mu.M, 10. Mu.M, 100. Mu.M, 1mM, 5mM, 10mM and detected using a sensor comprising a designed SPR sensor interface, respectively, and as a result, as shown in FIG. 3, the SPR angle was increased correspondingly with increasing concentration of the antigen to be detected, with a detection limit as low as 10nM and a linear range of 10nM to 10mM.
(2) Verification of sensor interface performance impact at common use temperatures
The novel coronavirus SARS-CoV-2 antigen concentration of 1 μm is detected by using the prepared SPR sensor interface for detecting SARS-CoV-2 antigen in aerosol at five temperatures of 25 ℃, 30 ℃, 35 ℃, 40 ℃ and 45 ℃ respectively, and as shown in FIG. 4, the temperature influence of the prepared SPR sensor interface at 25-45 ℃ is small, so that the detection error caused by temperature fluctuation in application can be reduced.
(3) Verification of interface stability for prepared SPR sensor:
the prepared SPR sensor interface for detecting SARS-CoV-2 antigen in aerosol is stored according to requirements, and standard samples with the concentration of the SARS-CoV-2 antigen of the same novel coronavirus of 1 mu M are detected on the 0 th day, the 5 th day, the 10 th day, the 15 th day, the 20 th day and the 30 th day, and the result is shown in figure 5, and the SPR angle of the detected same sample is only slightly reduced along with the increase of time, which indicates that the stability is good, and the performance is hardly changed within 30 days at room temperature.
Example 3
Different surface material thicknesses correspond to different SPR angle versus refractive index, and therefore, to verify the Cu employed 3 (PO 4 ) 2 The optimal thickness of the BSA-GO nano material is respectively prepared into Cu with five thicknesses of 10nm, 20nm, 30nm, 40nm and 50nm 3 (PO 4 ) 2 SPR sensor interface of BSA-GO layer for SARS-CoV-2 antigen detection in aerosol and Cu with different thickness prepared 3 (PO 4 ) 2 The sensor of the-BSA-GO layer detects the standard sample with the novel coronavirus SARS-CoV-2 antigen concentration of 1 mu M, and the result is shown in FIG. 6, no matter what thickness Cu is contained 3 (PO 4 ) 2 The surface of the BSA-GO layer has the lowest refractive index at about 55 DEG, but has a refractive index of almost 0 at the lowest part at a thickness of 30nm, and gradually increases with increasing thickness from 30nm, indicating that Cu with a thickness of 20nm to 40nm is selected 3 (PO 4 ) 2 The BSA-GO layer can achieve better detection effect as a designed SPR sensor interface, wherein Cu is as follows 3 (PO 4 ) 2 The thickness of the BSA-GO layer is optimally 30nm.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. An SPR sensor interface for detecting SARS-CoV-2 antigen is composed of high-refractive index glass, indium tin oxide layer, amorphous silicon layer, gold layer and Cu 3 (PO 4 ) 2 -BSA-GO nanoflower layer, cu 3 (PO 4 ) 2 the-BSA-GO nanoflower layer comprises Cu coated with ACE2 protein probes 3 (PO 4 ) 2 -BSA-GO nanoflower;
the Cu is 3 (PO 4 ) 2 The BSA-GO nanoflower is obtained by precipitation reaction of copper sulfate, bovine serum albumin and graphene oxide in phosphate buffer solution;
the refractive index of the high refractive index glass is 1.72-1.82;
the Cu is 3 (PO 4 ) 2 The thickness of the BSA-GO nano-flower layer is 20-40 nm.
2. The SPR sensor interface for detecting SARS-CoV-2 antigen according to claim 1, wherein the high refractive index glass has a thickness of 0.75 to 1mm;
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.
3. The SPR sensor interface for detecting SARS-CoV-2 antigen according to claim 1, wherein 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 ℃ and the time is 20-30 h.
4. The SPR sensor interface for detecting SARS-CoV-2 antigen according to claim 1, wherein said ACE2 protein is bound to Cu 3 (PO 4 ) 2 The mass ratio of the BSA-GO nanoflowers is 1:6-10.
5. The method for preparing an SPR sensor interface for detecting SARS-CoV-2 antigen as described in any one of claims 1 to 4, comprising the steps of:
sputtering indium tin oxide onto the surface of high refractive index glass by a first magnetron sputtering method to form an indium tin oxide layer;
performing second magnetron sputtering on amorphous silicon to the surface of the indium tin oxide layer to form an amorphous silicon layer;
sputtering gold on the surface of the amorphous silicon layer by a third magnetron sputtering method to form a gold layer;
ACE2 protein, cu 3 (PO 4 ) 2 Mixing the BSA-GO nanoflower with a buffer solution, and performing first incubation to obtain Cu coated with ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflower;
cu coated with ACE2 protein probe 3 (PO 4 ) 2 Mixing the BSA-GO nanoflower with bovine serum albumin, and performing second incubation to obtain the closed Cu coated with ACE2 protein probe 3 (PO 4 ) 2 -BSA-GO nanoflower;
cu coating the closed Cu coated with ACE2 protein probe 3 (PO 4 ) 2 And depositing BSA-GO nanoflower on the surface of the gold layer to obtain an SPR sensor interface for detecting SARS-CoV-2 antigen.
6. The method according to claim 5, wherein the first magnetron sputtering gas is helium, the sputtering pressure is 1-1.4 Pa, the sputtering power is 120-180W, the sputtering time is 3-5 min, and the sputtering vacuum degree is 6X 10 -4 Pa; the distance between the sputtering target source and the high refractive index glass is 8-12 cm.
7. The method according to claim 5, wherein the second magnetron sputtering gas is helium, the sputtering pressure is 2-3 Pa, the sputtering power is 150-250W, the sputtering time is 0.8-1.2 h, and the sputtering vacuum degree is 6X 10 -4 Pa; the distance between the sputtering target source and the indium tin oxide layer is 8-12 cm.
8. The method according to claim 5, wherein the third magnetron sputtering gas is helium, the sputtering pressure is 2.5-3.5 Pa, the sputtering power is 240-260W, the sputtering time is 30-50 s, and the sputtering vacuum degree is 6X 10 - 4 Pa; the distance between the sputtering target source and the amorphous silicon layer is 8-12 cm.
9. The method according to claim 5, wherein the first incubation is performed at room temperature for 2 to 3 hours; the temperature of the second incubation is room temperature and the time is 0.5-1.5 h.
10. A method for detecting SARS-CoV-2 antigen, comprising the steps of:
covering a sample to be detected with the SPR sensor interface for detecting SARS-CoV-2 antigen according to any one of claims 1-4 or the SPR sensor interface for detecting SARS-CoV-2 antigen prepared by the preparation method according to any one of claims 5-9, and incubating;
testing the SPR angle of the SPR sensor interface obtained after incubation, and obtaining the content of SARS-CoV-2 antigen in the solution to be tested according to the SPR angle and a preset standard curve;
the standard curve is a linear relationship curve of SPR angle and SARS-CoV-2 antigen concentration.
CN202310133712.XA 2023-02-20 2023-02-20 SPR sensor interface for detecting SARS-CoV-2 antigen and its detecting method Pending CN116106546A (en)

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