CN112730340B

CN112730340B - Optical fiber sensor for quickly detecting novel coronavirus SARS-CoV-2

Info

Publication number: CN112730340B
Application number: CN202110065903.8A
Authority: CN
Inventors: 段忆翔; 罗泽伟; 何露; 郭星
Original assignee: Northwest University
Current assignee: Northwest University
Priority date: 2021-01-19
Filing date: 2021-01-19
Publication date: 2022-03-01
Anticipated expiration: 2041-01-19
Also published as: CN112730340A

Abstract

An optical fiber sensor for detecting a novel coronavirus SARS-CoV-2, comprising: fiber probe, signal amplification probe and sensor platform. The sensor can be used for developing direct analysis method and sandwich method, both of which can be used for detecting SARS-CoV-2. The invention modifies the aptamer of the N protein of the targeting SARS-CoV-2, designs the T-shaped aptamer, and greatly improves the efficiency of capturing the N protein by the optical fiber probe. The direct analysis method can be developed by fixing the T-shaped nucleic acid aptamer on the surface of the optical fiber. The prepared gold/silver nanosphere-aptamer (Au/AgNPs-apt) is used for amplifying a sensor signal, and then a sandwich method can be developed for high-sensitivity detection of SARS-CoV-2. The sandwich method has double aptamers and can be used for enhancing the specificity of detection. The sandwich method has the advantages of high specificity, high sensitivity and high detection speed, and is an effective supplement for the detection of the nucleic acid of the new coronavirus.

Description

Optical fiber sensor for quickly detecting novel coronavirus SARS-CoV-2

Technical Field

The invention relates to the technical field of optical fiber sensors, in particular to preparation and application of optical fiber local surface plasma for detecting novel coronavirus SARS-CoV-2.

Background

The new coronavirus pneumonia (COVID-19) epidemic situation is outbreak worldwide, seriously harms human health and has great influence on public health safety. Although COVID-19 has a lower mortality rate than Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS), it is extremely contagious. COVID-19 mainly spreads respiratory droplets and spreads by human-borne means. COVID-19 is a disease caused by a novel coronavirus (SARS-CoV-2). SARS-CoV-2 is an RNA virus, which is composed mainly of S protein, N protein, spike protein and RNA. The virus has strong stability on the surface of refrigerated or frozen food and has infectivity. Recently, researchers in our country have found that COVID-19 can also be transmitted indirectly through cold chain foods. In order to ensure the safety of food and cut off the transmission path, the development of a simple, rapid and highly sensitive analysis method for diagnosing SARS-CoV-2 on the surface of cold chain food is urgently needed.

Currently, researchers have developed various diagnostic methods for SARS-CoV-2, mainly including real-time fluorescent quantitative PCR (qPCR), immunodipsticks, and enzyme-linked immunosorbent assay (ELISA). Although qPCR is used as the gold standard for codv-19 detection, it still has disadvantages in practical applications, including expensive instrumentation, time consuming analysis and cumbersome RNA pre-treatment steps. The immune test strip is a rapid detection method, and immune globulin Ig G and Ig M in a patient body are analyzed. ELISA is also an analytical method developed based on Ig G and Ig M in serum. However, the surface of cold chain foods is free of immunoglobulins Ig G and Ig M. Therefore, the direct analysis method of SARS-CoV-2 is developed, which can be used for the on-site rapid analysis of cold chain food and has important significance for ensuring the food safety.

Fiber Localized Surface Plasmon Resonance (LSPR) sensors are a real-time label-free analytical technique. The sensor has the advantages of simple optical path, easy integration and miniaturization. Omega-shaped fibers have a higher refractive sensitivity than U-shaped and straight fibers. The omega-shaped optical fiber LSPR sensor established by the method can realize sensitive and rapid analysis of SARS-CoV-2 on the surface of food, and is expected to become a beneficial supplement for nucleic acid analysis.

Disclosure of Invention

In order to overcome the problem of time consumption of detection in the prior art, the invention aims to provide the omega-shaped optical fiber sensor for directly analyzing SARS-CoV-2, the preparation method and the application of the sensor, and realize the SARS-CoV-2 analysis on the surface of cold chain food. The present invention directly analyzes SARS-CoV-2 specific protein. Compared with the method based on the virus RNA analysis, the method does not need additional sample pretreatment and can directly carry out detection and analysis. The omega-shaped optical fiber constructed by the invention has high refraction sensitivity, and the optical fiber sensor constructed by the invention has bright characteristics in the aspects of on-site, rapid and high-sensitivity detection and the like.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides an omega-shaped local surface plasmon resonance optical fiber sensor, which can realize two modes of detection and analysis methods: direct assay and sandwich methods. The direct analysis method comprises an omega-shaped optical fiber probe and an optical fiber sensor platform, and the sandwich method comprises the omega-shaped optical fiber probe, a signal amplification probe and the optical fiber sensor platform. The omega-shaped optical fiber probe comprises an omega-shaped optical fiber, nanogold and a T-shaped nucleic acid adapter; the signal amplification probe comprises core-shell structure Au/AgNPs-apt nanoparticles. The optical fiber sensor platform comprises a light source, a continuous spectrometer, software and an optical connecting part;

the local surface plasma resonance optical fiber sensor of the invention comprises: omega-shaped fiber probes and signal amplification probes. The invention also comprises a novel coronavirus SARS-CoV-2 detection method based on two modes of sensor development: direct assay and one-step sandwich. The invention modifies the aptamer of the targeted novel coronavirus N protein, prepares the T-shaped aptamer, improves the efficiency of capturing the N protein by the omega-shaped optical fiber probe, and establishes a direct analysis method aiming at the novel coronavirus SARS-CoV-2. The invention respectively fixes two T-shaped nucleic acid aptamers with different binding sites on the surfaces of the omega-shaped optical fiber probe and the core-shell structure Au/Ag nanosphere. As a signal amplification probe, the Au/Ag nanospheres with the core-shell structure and virus specific protein can form a sandwich structure, and a one-step sandwich method is established. The method can realize simple, rapid and high-sensitivity analysis of the novel coronavirus SARS-CoV-2. The optical fiber local surface plasma sensor has important application prospect in rapid analysis of the new coronavirus on the surface of the cold chain food, and can be used as beneficial supplement of clinical nucleic acid detection.

An omega-shaped optical fiber probe, the probe structure characteristics are as follows (see fig. 1 and fig. 10):

1) the cladding part comprises a naked part 1 and a cladding-containing part 2, wherein the naked part 1 comprises a circular arc section a larger than a semicircle and two straight sections b. Two ends of the arc section a are respectively connected with the straight section b, and the other end of the straight section b is connected with the cladding containing part 2.

2) Wherein the bare portion 1 contains only a core portion of the optical fiber and does not contain a coating layer and a cladding layer; the cladding-containing portion 2 contains a core, a coating layer and a cladding. Wherein the exposed part 1 is the sensing area.

3) The range of the diameter of the optical fiber core is 100-600 mu m, and the optimal range is 600 mu m; the length is 10-40 cm, preferably 35 cm. The length of the exposed part 1 can be selected to be 2-5 cm, preferably 4 cm.

Further, the omega-shaped optical fiber probe is prepared by the following steps:

1) taking an optical fiber, and burning off a cladding of a part needing to be exposed under the condition of high-temperature flame; then, properly bending the exposed part on the flame, and processing and forming; and finally, simultaneously burning out the cladding and the coating of about 2 cm at two ends of the optical fiber, and polishing the optical fiber to be smooth to prepare the omega-shaped optical fiber.

2) Immersing the sensing part of the omega-shaped optical fiber into the goby liquid (30% H)₂O₂:H₂SO₄3:7 v/v), reacting in an oven at 80 ℃ for 10-40 min, wherein the reaction time is preferably 30 min. And after the reaction is finished, ultrasonically washing the mixture, and drying the mixture in a drying oven for later use.

3) Soaking the omega-shaped optical fiber sensing part into 0.1% APTMS solution, wherein the solvent of APTMS is a mixed solution of ethanol and acetic acid (5: 2 v/v), treating for 15 min, and ultrasonically cleaning for later use.

4) The synthesized nano gold has the optional range of 13-50 nm, preferably 15 nm. And (3) immersing the processed omega-shaped optical fiber into a nano-gold solution, and modifying for 2.5-80 min, wherein the soaking time is preferably 60 min. And taking out the optical fiber oven for drying for later use.

5) 2 μ M of the aptamer in T form was annealed in a PCR instrument. After completion, NaCl solution was added to make Na⁺The final concentration of (3) was 0.3M. And (3) immersing the optical fiber sensing part modified by the nano-gold into a T-shaped nucleic acid aptamer solution for overnight treatment. Wherein the buffer solution is 99% PBS (pH 7.0-7.4) and 1% ionic solution (100 mM CaCl)₂And 50 mM MgCl₂Mixed liquid). After the reaction was completed, the omega-shaped optical fiber was washed with deionized water to remove unbound T-shaped nucleic acid aptamers. And then, placing the optical fiber in a glycine buffer solution or a BSA buffer solution for blocking for 15-120 min to obtain the omega-shaped optical fiber probe.

Further, the nanogold is obtained by a method comprising the following steps: 50 mL HAuCl in flask₄•3H₂And heating the O aqueous solution to boil. Different volumes of 1% trisodium citrate solutions were added to the flasks to synthesize nanogold with different particle size sizes. When the solution is wine red, boiling the mixture for 15 min, and cooling to room temperature under stirring.

The omega-shaped optical fiber probe can be used for constructing an optical fiber sensor, wherein the omega-shaped optical fiber probe needs to be modified with a T-shaped nucleic acid aptamer. The characteristics of the T-shaped nucleic acid fit are as follows:

1) the aforementioned T-shaped nucleic acid aptamers comprise two pieces of DNA, DNA1 and DNA 2. Wherein DNA1 comprises three domains, namely a ploy a region, a complementary region, and an aptamer region; DNA2 contains two domains, a ploy a region and a complementary region.

2) The 3 'or 5' end modifying group of the DNA1 and the DNA2 in the T-shaped nucleic acid aptamer can be selected from sulfydryl, poly A or streptavidin, and is preferably poly A.

3) The aforementioned T-shaped nucleic acid aptamers, wherein the length of the ploy A region in DNA1 can be selected in the range of 5A-50A, preferably 5A; the length of ploy A region in DNA2 can be selected from the range of 5A-50A, preferably 15A, and the aptamer region is an aptamer specifically targeting N protein.

4) The T-shaped nucleic acid aptamer, wherein the complementary sequences of DNA1 and DNA2 are random sequences, optionally 10-50 bp in length, preferably 15 bp in length. DNA1 and DNA2 were artificially synthesized by chemical methods. Preferred DNA1 and DNA2 sequences are as follows:

DNA 1：

5’-GCTGGATGTCGCTTACGACAATATTCCTTAGGGGCACCGCTACATTGACACATCCAGCgGAGGTAATAGTACGAAAAA- 3’

DNA 2：5’-CCTCCATTATCATGCAAAAAAAAAAAAAAA- 3’；

the fiber sensor developed to detect SARS-CoV-2 has two modes: direct assay and sandwich methods.

Further, the steps of modifying the nano-gold on the surface of the optical fiber are specifically realized as follows:

1) immersing the sensing part of the optical fiber in the goby liquid (30% H)₂O₂:H₂SO₄3:7 v/v), reacting in an oven at 80 ℃ for 30 min, after the reaction is finished, ultrasonically cleaning the optical fiber, and drying in the oven.

2) Soaking the optical fiber in 0.1% APTMS solution for 15 min, and ultrasonically cleaning with ethanol-acetic acid (5: 2 v/v) mixture.

3) The synthesis method of the nano gold comprises the following steps: 50 mL of HAuCl was added to the flask₄.3H₂And heating the O aqueous solution to boil. Different volumes of 1% trisodium citrate solution were added to the flask rapidly to synthesize nanogold with different particle size. When the solution turns wine red and is stable, the mixture is boiled for 15 min and cooled to room temperature under stirring.

4) And (3) immersing the treated optical fiber into a nano gold solution, and modifying for 2.5-80 min, wherein the optimal time is 60 min.

Further, modifying the aptamer on the surface of the omega-shaped optical fiber to prepare the optical fiber probe, which comprises the following specific steps:

1) the aptamers were prepared to the desired concentration, and annealed in a buffer solution of 99% PBS plus 1% ionic solution (100 mM calcium chloride and 50 mM magnesium chloride mixed solution). After the annealing was completed, a sodium chloride solution was added to make the final concentration 0.3M.

2) And (3) immersing the optical fiber sensing part modified by the nano-gold into the treated aptamer solution, and connecting the aptamer after night.

The operation steps of the direct analysis method are as follows:

soaking the omega-shaped optical fiber probe modified with the T-shaped nucleic acid aptamer in a binding buffer solution, and balancing for 1-60 min, preferably 10 min. Then, N protein specific to SARS-CoV-2 was added, and changes in Local Surface Plasmon Resonance (LSPR) absorption peak values were recorded in real time. And establishing a method for detecting the N protein according to the change of the LSPR absorption peak value.

The sandwich method comprises the following operation steps:

soaking the omega-shaped optical fiber probe modified with the T-shaped nucleic acid aptamer in a binding buffer solution, adding a certain volume of 1 nM Au/AgNPs-apt, and balancing for 1-60 min, preferably 20 min. Then, N protein specific to SARS-CoV-2 was added, and changes in Local Surface Plasmon Resonance (LSPR) absorption peak values were recorded in real time. And establishing a method for detecting the N protein according to the change of the LSPR absorption peak value. The optional volume of Au/AgNPs-apt is 50-300. mu.L, preferably 200. mu.L.

Incidentally, the sandwich method requires a signal amplification probe. The signal amplification probe can be selected from a nano gold-aptamer, a nano silver-aptamer or a nano gold/silver-aptamer (Au/AgNPs-apt), and is preferably Au/AgNPs-apt.

Further, the preparation method of the core-shell structure Au/AgNPs comprises the following steps:

1) 1mL of 16 nm citrate reduced Au seeds were transferred to a 1.5 mL centrifuge tube, 14 uL of 0.1M silver nitrate solution was added, and vortex 10S mixed.

2) Equal volumes of 0.1M ascorbic acid and 2 uL of 25% aqueous ammonia were added and the mixture was vortexed for 20S and placed in a 4 ℃ freezer for further use.

The preparation method of Au/AgNPs-apt is as follows:

1) 1mL of 16 nm citrate-reduced AuNPs were used as seeds, transferred to a 1.5 mL centrifuge tube, and 14. mu.L of 0.1M AgNO was added₃Solution, mix vortex 10 s. The AuNPs seeds can have a particle size of 13-50 nm, preferably 20 nm.

2) Adding 0.1M ascorbic acid and 2 mu L25% ammonia water solution with equal volume, and vortexing the mixture for 20 s to obtain the product Au/AgNPs with the core-shell structure, and putting the product into a refrigerator at 4 ℃ for later use.

3) mu.M of DNA1 and DNA3 were mixed in PBS buffer and co-annealed, respectively. And dropwise adding the annealed product into the Au/Ag nanospheres with the core-shell structures. After standing for 3 min, citric acid (pH 3.0, 500 mM) was added dropwise for 20 min. Centrifuging twice to obtain a precipitate of Au/AgNPs-apt, namely a signal amplification probe.

The aforementioned fiber optic sensor, DNA3, is structurally similar to DNA1 described above: i.e., comprising a ploy A region, a complementary region, and an aptamer region. The DNA3 is artificially synthesized by a chemical method. The preferred DNA3 sequence is:

5’-GCTGGATGTCACCGGATTGTCGGACATCGGATTGTCTGAGTCATATGACACATCCAGCgGAGGTAATAGTACGAAAAA-3’。

an optical fiber sensor for detecting SARS-CoV-2. The sensor comprises an optical fiber probe, a signal amplification probe and a sensor platform. The surface of the optical fiber probe is modified with nanogold and a T-shaped nucleic acid aptamer; the signal amplification probe is Au/AgNPs-apt.

Further, the equipment required for building the platform comprises: the optical fiber probe, the continuous spectrometer, the tungsten lamp light source, the computer and the optical connecting component. The nano-gold with different OD values is modified on the optical fiber sensing part, and the refraction sensitivity is different.

The sensor platform comprises a compact fiber spectrometer, a light source, a displacement table, a sample cell and a constant-temperature metal bath. The light source can be selected from an LED lamp light source or a halogen tungsten lamp light source, and is preferably a stable halogen tungsten lamp light source. The sample cell can be selected as an autonomously processed sample cell or a centrifugal tube, and is preferably a centrifugal tube; the volume of the sample cell can be selected to be 0.2-10 mL, and is preferably a centrifuge tube of 0.6 mL.

The combination conditions when the optical fiber captures the N protein in the optical fiber sensor are as follows: the temperature is 25 ℃ and the buffer is 99% PBS plus 1% ionic solution (100 mM calcium chloride and 50 mM magnesium chloride mixture). This condition is applicable to both the direct assay and the sandwich method.

The invention has proved that the specificity verification substance of the N protein is ovalbumin, bovine serum albumin, concanavalin A, compound protease and alpha-amylase. Wherein the concentration of said N protein varies from 0.001. mu.g/mL to 5. mu.g/mL. Ovalbumin, bovine serum albumin, concanavalin A, compound protease and alpha-amylase were used as controls for specific recognition, and the concentration was 25. mu.g/mL.

The optical fiber local surface plasma resonance probe sensor, wherein the protein and the aptamer are combined and dynamically monitored, and the preferred monitoring time is 120 min.

The optical fiber sensor can realize the rapid analysis of SARS-CoV-2 on the surface of cold chain food. The assay method may be selected as a direct assay or a sandwich method, preferably a sandwich method. The monitoring time is 15-120 min, preferably 15 min. The preferred analytical procedure is as follows:

1) and dipping the sterilized cotton ball with the binding buffer solution, and wiping the surfaces of the cold chain food shrimps or the packaging boxes for three times respectively. The cotton ball was centrifuged and the wiping solution was collected.

2) And adding wiping liquid after the reaction of the optical fiber sensor and the signal amplification probe is balanced. And collecting characteristic absorption peaks, and calculating the N protein content on the surface of the shrimp or the packaging box.

3) And adding the collected wiping solution into a standard amount of N protein to prepare a simulated positive sample. And (5) measuring the content of the positive sample and calculating the recovery rate.

The protein detected by the optical fiber sensor is N protein of SARS-CoV-2. By replacing the nucleic acid aptamer sequences in DNA1 and DNA3, the present sensor can be used to analyze the S, M, and E proteins of SARS-CoV-2.

In particular, the application of the optical fiber sensor in detecting epidemic diseases. The application can be selected from the surface of cold chain food and packaging boxes, but is not limited to the surface of the cold chain food and packaging boxes. The application steps are as follows:

1) and dipping the sterilized cotton balls in the combined buffer solution, and wiping the surfaces of the cold chain food and the packaging box. The wiping solution was collected. Adding the wiping solution into an optical fiber sensor, detecting for 15 min in real time, recording the change of characteristic absorption peaks by a sensor platform, and calculating the N protein content on the surfaces of cold chain food and packaging boxes.

2) A positive sample for SARS-CoV-2 simulation was prepared by adding a standard amount of N protein to the swab. And (3) adding the positive sample into the optical fiber sensor, detecting for 15 min in real time, recording the change of the characteristic absorption peak by a sensor platform, and calculating the recovery rate of the detection method.

Further, the N protein concentration was varied from 0.001. mu.g/mL to 5. mu.g/mL. Ovalbumin, bovine serum albumin, concanavalin A, compound protease and alpha-amylase were used as controls for specific recognition, and the concentration was 25. mu.g/mL.

The binding of the protein to the aptamer was monitored dynamically for 1 h.

The binding conditions of the protein and the aptamer are as follows: the temperature was 25 ℃ and the buffer was 99% PBS plus 1% ionic solution (100 mM CaCl)₂And 50 mM MgCl₂Mixed liquid).

The invention has the beneficial effects that:

compared with the existing SARS-CoV-2 detection method, the optical fiber sensor for rapidly detecting SARS-CoV-2 provided by the invention has obvious advantages, which are as follows:

1. the omega-shaped optical fiber sensor can detect SARS-CoV-2 in real time without marks and with high sensitivity. Compared with a PCR detection method, the detection method is simple to operate, does not need sample pretreatment, can realize direct analysis by a one-step method, and has short detection time (the shortest time is 15 min). The invention utilizes the Au/AgNPs-apt nano material with the core-shell structure to amplify signals, has the sensitivity characteristic of time dependence and can realize sensitive analysis.

2. The invention has two N protein modes for detecting SARS-CoV-2: direct assay and sandwich methods. Both methods allow for rapid analysis (15 min), both with time-dependent sensitivity characteristics. The two detection methods can provide different linear ranges, and the application range of the optical fiber sensor is widened.

3. The present invention designs a new T-shaped nucleic acid aptamer which can improve the speed and the sensitivity of detecting SARS-CoV-2. Compared with the nucleic acid aptamer modified by sulfydryl and ploy A, the T-shaped nucleic acid aptamer can more effectively regulate the density and the molecular state of the T-shaped nucleic acid aptamer on the surface of the optical fiber probe. Therefore, the omega-shaped optical fiber probe can effectively capture the reaction kinetics of the N protein.

4. The double-nucleic-acid aptamer adopted by the invention can improve the specificity of detection and greatly reduce the false positive of detection.

5. The omega-shaped optical fiber sensor device has small volume and is easy to integrate. The detection process can be carried out at room temperature, no additional instrument is needed, and the requirement of SARS-CoV-2 field detection can be met.

6. The detection principle adopted by the invention is that the aptamer connected on the optical fiber is specifically combined with the substance to be detected, and has high specificity and affinity to the substance to be detected. Meanwhile, the sample does not need to be marked, and the damage and the pollution to the sample are reduced.

7. The invention can monitor the reaction process of the sample in real time and can truly reflect the interaction process of the N protein and the aptamer.

Drawings

FIG. 1 is a schematic diagram of an optical fiber sensor for detecting N protein. (a) Experimental procedures of direct assay and sandwich; (b) an optical fiber object graph; (c) composition of the aptamer of the T-form.

Fig. 2 is a fiber optic sensor platform.

FIG. 3 is a timing diagram of the detection of N protein by the direct assay. The concentrations of N protein were 1, 2, 20, 50, 100 and 200 nM, respectively.

FIG. 4 is a standard curve for detecting N protein by direct assay. Under the conditions that the detection time is 15 min, 30 min, 45 min and 60 min, the optical fiber sensor can obtain good linear relation.

FIG. 5 is a specificity assay. A to F are respectively: 2.5. mu.g/mL N protein, 25. mu.g/mL ovalbumin, 25. mu.g/mL bovine serum albumin, 25. mu.g/mL concanavalin A, 25. mu.g/mL complex protease, and 25. mu.g/mL alpha-amylase.

FIG. 6 shows the synthesis of signal amplification probe Au/AgNPs-apt. (a) Ultraviolet absorption spectrograms of AuNPs, Au/AgNPs and Au/AgNPs-apt. (a inset diagram) real object diagrams of AuNPs (1), Au/AgNPs (2) and Au/AgNPs-apt (3). (b) Transmission electron microscopy images and elemental distribution of Au/AgNPs.

FIG. 7 is a transmission electron microscope image of a signal amplification probe Au/AgNPs-apt.

FIG. 8 is a timing diagram of detection of N protein by the sandwich method. The concentrations of N protein were 0.02, 0.2, 1, 2, 10 and 20 nM, respectively.

FIG. 9 is a standard curve for detecting N protein by the sandwich method. The optical fiber sensor can obtain good linear relation under the conditions that the detection time is 15 min, 30 min, 45 min, 60 min, 90 min and 120 min.

FIG. 10 is a schematic view of the structure of the omega-shaped optical fiber probe of the present invention.

In the figure, 1-optical fiber bare part, 101-straight section, 102-bending section, 2-optical fiber containing cladding part, 3-core-shell structure Au/AgNPs-apt nanosphere, 4-light source, 5-sample cell, 6-spectrometer and 7-computer.

Detailed Description

The invention will be further elucidated with reference to the drawings and embodiments, but is not limited to the embodiments described below.

Example 1

A preparation method of an omega-shaped optical fiber probe comprises the following specific implementation method:

1) an omega-shaped optical fiber was prepared. And taking the optical fiber, burning off a cladding of the sensing part under the condition of high-temperature flame, and making the sensing part into an omega shape under the condition of high-temperature flame. While removing approximately 2 cm of cladding at both ends and smoothing (see fig. 1).

2) Preparing an omega-shaped optical fiber probe. And modifying the nano-gold on the surface of the optical fiber by using monolayer amino silane. Subsequently, DNA1 and DNA2 were diluted to 2. mu.M with phosphate buffer (pH 7.0-7.4) and annealed in a PCR instrument. The nanogold-modified optical fiber was immersed in the mixture of DNA1 and DNA2, and sodium chloride was added to a final concentration of 0.3 mM, and treated overnight at room temperature. The prepared omega-shaped optical fiber probe was stored at 4 ℃ for further use.

3) And (5) building an optical fiber sensor platform. The light source, the spectrometer, the software, the displacement table, the optical connecting part and the like are organically assembled (see fig. 2). And adjusting a light path by adopting ultrapure water, and using sucrose solutions with different concentrations (0-20 percent, mass/volume ratio) for calculating the refraction sensitivity of the omega-shaped optical fiber probe.

Example 2

The development of direct analysis method for detecting SARS-CoV-2 includes the following steps:

the omega-shaped fiber probe was placed in a 1.5 mL centrifuge tube, 540. mu.L of binding buffer was added, and equilibration treatment was performed at room temperature for 5 min. 60 μ L of N protein was added at various concentrations, with the concentration of N protein ranging from 1-200 nM. The fiber spectrometer monitors and records the signal change of the characteristic absorption peak in real time, and the signal change can reflect the combination condition of the N protein and the T-shaped nucleic acid aptamer in real time. The absorption value (A) at the absorption peak is taken₅₅₀) A timing chart and a standard curve are made (see fig. 3 and 4). The detection limit of the method for detecting the N protein in the SARS-CoV-2 is calculated.

Example 3

The specificity verification experiment of the direct analysis method comprises the following specific steps:

single protein specificity verification: the fiber was placed in a 1.5 mL centrifuge tube, 540. mu.L of buffer was added, and equilibration was performed at room temperature for 5 min. Adding ovalbumin, bovine serum albumin, concanavalin A, compound protease and alpha-amylase, adding one protein each time, wherein the final concentration is 25 mu g/mL, and monitoring the signal change condition of an absorption peak in real time.

And (3) verifying the specificity of the mixed protein: the fiber was placed in a 1.5 mL centrifuge tube, 540. mu.L of buffer was added, and equilibration was performed at room temperature for 5 min. Mixing the five proteins with N protein (the mass fraction concentration ratio is 10: 1), adding into a centrifuge tube, and measuring FOLSPR light absorption value signals. And (3) measuring FOLSPR absorbance signals after three proteins are mixed with the N protein and two proteins are mixed with the N protein. The absorbance at the peak of absorption was plotted against time (see FIG. 6) to reflect the binding of the aptamer to the protein in real time.

Example 4

The preparation of the signal amplification probe Au/AgNPs-apt nanosphere is specifically explained as follows:

1) synthesizing Au/Ag nanospheres. 1mL of 16 nM AuNPs (20 nM particle size) was transferred to a 1.5 mL centrifuge tube, 14. mu.L of 0.1M silver nitrate solution was added, and vortex was mixed for 10 s. mu.L of 0.1M ascorbic acid and 2. mu.L of 25% ammonia solution were added, and the mixture was vortexed for 20 seconds to prepare Au/Ag nanospheres.

2) And synthesizing the Au/AgNPs-apt nanosphere. DNA2 and DNA3 were mixed in sodium nitrate buffer and placed in a PCR machine for annealing. Subsequently, the mixture of DNA2 and DNA3 was added drop-wise to the Au/Ag nanospheres and allowed to stand for 3 min. Citric acid (pH 3.0, 500 mM) was added dropwise for 20 min. Centrifuging twice to obtain the precipitate as Au/AgNPs-apt nanosphere, namely the signal amplification probe. Redissolving with sodium nitrate solution, and storing at room temperature. And characterizing the synthesized Au/AgNPs-apt nanosphere by adopting an ultraviolet and transmission electron microscope.

Example 5

The sandwich method based on omega-shaped optical fiber sensor comprises the following steps of:

add 1% ionic solution (100 mM CaCl) to 99% PBS₂And 50 mM MgCl₂Mixed solution) to dilute the synthesized signal amplification probe Au/AgNPs-apt nanosphere to the desired concentration. The fiber was placed in a 1.5 mL centrifuge tube, 540. mu.L of diluted Au/AgNPs-apt nanosphere solution was added, and equilibration treatment was performed at room temperature for 20 min. Different amounts of 0.25 mg/mL N protein were added for real-time monitoringSignal change of characteristic absorption peak (see fig. 6). The absorption value at the absorption peak is taken to make a time curve and standard curves with different concentrations, so that the combination condition of the protein and the aptamer can be reflected in real time, and the detection amplification capacity of the signal amplification probe can be measured. Realizes real-time, label-free and sensitive detection of the novel coronavirus N protein.

Example 6

An experiment for rapidly detecting N protein in the surface of frozen shrimps and a packaging box SARS-CoV-2 by a sandwich method comprises the following specific steps:

1) and (3) actual sample detection: the sterilized cotton balls were dipped in the binding buffer, and the frozen shrimps and the surfaces of the packaging box were wiped with different cotton balls with forceps. The surface of each shrimp was wiped three times with the same cotton ball and the surface of the package box was also wiped three times. The wiped cotton balls were placed in 1.5 mL centrifuge tubes, respectively. And centrifuging by a mini centrifuge, and separating and collecting the wiping solution in the cotton balls. The omega-shaped fiber probe was placed in the binding buffer containing the Au/AgNPs-apt nanospheres and equilibrated for a period of time. And adding 60 mu L of wiping solution into a centrifugal tube in an optical fiber sensing platform, and recording the characteristic absorption peak in real time by a spectrometer for 15 min. The N protein content of the frozen shrimp and the package surface was calculated according to the standard curve of the sandwich method (see Table 1).

2) And (3) standard addition recovery test: wiping solutions for wiping the surfaces of shrimps and the packaging box are respectively collected, and N protein with standard concentration is added to simulate a positive sample infected by SARS-CoV-2. And adding 60 mu L of wiping solution into a centrifugal tube in the optical fiber sensing platform, and recording the characteristic absorption peak in real time by a spectrometer for 15 min. The recovery of N protein from the surface of the test frozen shrimps and the package boxes was calculated according to the standard curve of the sandwich method (see Table 1).

TABLE 1 Sandwich method for rapid analysis of N protein in surface SARS-CoV-2 of frozen shrimp and packaging box

Note: represents no N protein detected.

Table 1 shows the results of sandwich method for rapid analysis of N protein in SARS-CoV-2 on the surface of frozen shrimp and packaging box.

Sequence listing

<110> northwest university

<120> an optical fiber sensor for rapid detection of novel coronavirus SARS-CoV-2

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 78

<212> DNA

<213> Artificial Sequence

<400> 1

gctggatgtc gcttacgaca atattcctta ggggcaccgc tacattgaca catccagcgg 60

aggtaatagt acgaaaaa 78

<210> 2

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 2

cctccattat catgcaaaaa aaaaaaaaaa 30

<210> 3

<211> 78

<212> DNA

<213> Artificial Sequence

<400> 3

gctggatgtc accggattgt cggacatcgg attgtctgag tcatatgaca catccagcgg 60

aggtaatagt acgaaaaa 78

Claims

1. An optical fiber probe for detecting SARS-CoV-2, comprising a bare portion and a portion containing a cladding, the bare portion being connected to the portion containing the cladding; the naked part is a sensing area, the optical fiber probe only has a fiber core of an optical fiber at the naked part, and the surface of the fiber core is modified with a nano gold nucleic acid aptamer, a nano silver nucleic acid aptamer and/or a gold/silver nanosphere nucleic acid aptamer with a core-shell structure; the sensing area of the optical fiber probe comprises a straight section and a bending section, and an optical fiber core in the bending section is in an omega shape;

the aptamer is a T-shaped aptamer and comprises DNA1 and DNA 2;

the base sequence of the DNA1 is: 5'-GCTGGATGTCGCTTACGACAATATTCCTTAGGGGCACCGCTACATTGACACATCCAGCgGAGGTAATAGTACGAAAAA-3', respectively;

the base sequence of the DNA2 is: 5'-CCTCCATTATCATGCAAAAAAAAAAAAAAA-3' are provided.

2. The fiber probe of claim 1, wherein the surface of the core is modified with spherical nanogold of 13-50 nm; the DNA1 is complementary with a complementary region of the DNA2, is a random sequence and has the length of 10-50 bp; DNA1 contains an aptamer region, a complementary region, and a ploy a region targeted to the new coronavirus N protein; DNA2 contains a ploy A region and a complementary region.

3. The fiber probe of claim 1, wherein the fiber has a core diameter of 100-600 μm and a length of 10-40 cm; the length of the exposed part ranges from 2 cm to 5 cm.

4. A method for preparing the optical fiber probe according to any one of claims 1 to 3, comprising the steps of:

1) taking an optical fiber, removing a cladding of a sensing part under the condition of high-temperature flame, processing the optical fiber into a required shape on the flame, removing the cladding with the length of about 2 cm at two ends, and polishing the optical fiber smoothly;

2) modifying nano-gold on the surface of the optical fiber by using monomolecular-layer aminosilane; subsequently, the optical fiber was immersed in phosphate buffer of pH 7.0 to 7.4 of annealed DNA1 and DNA 2; adding sodium chloride to a final concentration of 0.3M, and treating at room temperature overnight; storing at 4 deg.C for use.

5. The signal amplification probe used in combination with the optical fiber probe according to any one of claims 1 to 3, wherein the signal amplification probe is a nanogold nucleic acid aptamer, a nanogold nucleic acid aptamer and/or a core-shell structure gold/silver nanosphere nucleic acid aptamer; the aptamer is a T-shaped aptamer and comprises DNA2 and DNA 3;

the base sequence of the DNA2 is: 5'-CCTCCATTATCATGCAAAAAAAAAAAAAAA-3', respectively;

the base sequence of the DNA3 is: 5'-GCTGGATGTCACCGGATTGTCGGACATCGGATTGTCTGAGTCATATGACACATCCAGCgGAGGTAATAGTACGAAAAA-3' are provided.

6. The signal amplification probe of claim 5, wherein the DNA3 comprises an aptamer region, a complementary region, and a ploy A region targeted to the N protein of a novel coronavirus.

7. The method for preparing a signal amplification probe according to claim 5 or 6, comprising the steps of: mixing DNA2 and DNA3 in phosphate buffer and/or sodium nitrate solution for annealing, and then dropwise adding into nano gold, nano silver and/or core-shell structure gold/silver nanospheres; standing for 3 min, adding 500 mM citric acid with pH of 3.0 dropwise, and reacting for 20 min; centrifuging twice, and obtaining a precipitate as a signal amplification probe.