Application of a bionic heparan sulfate AIE fluorescent probe in detecting SARS-CoV-2 virus

Abstract

The present invention relates to the technical field of virus detection, and discloses an application of an AIE fluorescent probe of bionic heparan sulfate in detecting SARS-CoV-2 virus. The structural formula of the AIE fluorescent probe is as follows. The fluorescent probe is a water-soluble probe, which can adhere to the periphery of the virus after binding to the Spike protein on the surface of the coronavirus, so as to induce aggregation and emit light. After the novel coronavirus or protein is incubated, the fluorescence intensity of the fluorescent probe increases by nearly 20 times, and the probe has the advantages of high sensitivity, high resolution, low background noise and high stability. At the same time, the adhesion of the fluorescent probe to the virus occurs instantly, and the accurate value of the virus titer can be obtained within 15 seconds, so that a rapid detection effect can be achieved, and simultaneous screening and testing of large quantities of samples can be satisfied.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and in particular to an application of a bionic heparan sulfate AIE fluorescent probe in detecting SARS-CoV-2 virus. Background Art

[0002] The main transmission routes of the virus are respiratory droplets and close contact. Contact with virus-contaminated objects can also cause infection, and exposure to aerosols with high concentrations of the virus can also cause infection. Many virus carriers do not have any relevant symptoms in the early stages, making it difficult to be identified, which prevents the widespread spread of the virus. In order to curb the spread of SARS-CoV-2 as soon as possible, it is necessary to develop rapid and accurate virus detection technology.

[0003] At present, the detection of COVID-19 mainly targets viral nucleic acid, viral antigen and viral immunology. Nucleic acid detection uses polymerase chain reaction technology (PCR) or DNA-RNA molecular hybridization to detect SARS-CoV-2 RNA. Antigen detection detects whether there is viral protein in the saliva, nasal or nasopharyngeal swabs or even blood of infected people. Immunological detection is an indirect method of detecting viruses. It detects not the virus itself, but the antibodies produced in the body of the infected person. Nucleic acid detection has the characteristics of high sensitivity and high specificity, and is considered to be the "gold standard" for early diagnosis of SARS-CoV-2, but it is expensive and requires high technical skills from the operator; in addition, the sensitivity of existing antigen and antibody detection methods is limited and it takes hours to get results. Therefore, it is necessary to develop simpler, faster, more sensitive and accurate detection methods.

[0004] Fluorescence detection has the advantages of high sensitivity, high resolution, fast response, low background noise, and simple operation, and has attracted widespread attention. Due to its unique photoelectric properties and biological activity, fluorescent probes have been used in clinical detection and scientific research of various biological molecules. Since Academician Tang Benzhong proposed the aggregation-induced emission (AIE) effect in 2001, molecules with the AIE effect have been named AIEgen (molecules with AIE properties). In contrast to the aggregation fluorescence quenching effect, AIEgen emits weakly in dilute solutions and has enhanced fluorescence in the aggregated state.

[0005] For example, (CN 110606859 A) discloses an aggregation-induced emission compound, which utilizes the aggregation-induced emission effect of AIE molecules, and is used in a detection method and a related kit that integrates two signal modes, fluorescence and plasma colorimetry, for detecting various immune-related target analytes. This proves that aggregation-induced emission compounds are of great significance in the field of biological detection and have broad application prospects.

[0006] For example, (CN 111239391 A) discloses a novel coronavirus antigen detection reagent and detection device, and designs a novel coronavirus antigen detection reagent with AIE nanospheres, the core of which is to use the novel coronavirus N protein antibody as a monoclonal antibody to specifically bind to the virus. This is similar to the antigen test kits available on the market, and the use of monoclonal antibodies will increase the cost of virus detection.

[0007] Due to the problems that existing novel coronavirus testing is inconvenient, requires high operators, is expensive, and time-consuming, there is an urgent need for a highly sensitive and fast fluorescent detection method for the novel coronavirus. Summary of the invention

[0008] In view of the problems that existing novel coronavirus detection is inconvenient and time-consuming, the present invention provides an AIE fluorescence detection method for SARS-CoV-2 virus, which has the advantages of high sensitivity, high resolution, and low background noise, is suitable for simultaneous detection of large quantities of samples, and has low preparation cost, simplicity and speed.

[0009] To achieve the above object, the technical solution adopted by the present invention is:

[0010] Application of a bionic heparan sulfate AIE fluorescent probe in detecting SARS-CoV-2 virus, the structural formula of the AIE fluorescent probe (abbreviated as TPES) is as follows:

[0011]

[0012] The present invention utilizes the combination of coronavirus and heparan sulfate to discover an AIE fluorescent probe that mimics heparan sulfate proteoglycan. The sulfonic acid group is modified on (tetra-(4-hydroxyphenyl)ethylene) TPE to obtain a new AIEgen molecule, referred to as TPES. The structure of TPES is similar to that of heparan sulfate proteoglycan, so it can bind to the Spike protein on the surface of the coronavirus and adhere to the virus, so as to induce aggregation and emit light. After incubation with the new coronavirus or protein, the fluorescence intensity of the fluorescent probe increases by nearly 20 times, which has a very obvious change effect, and can realize rapid qualitative and quantitative detection of new tubular viruses.

[0013] The SARS-CoV-2 virus includes any one or more of the Spike proteins of the mutant Omicron, the mutant Delta, the wild species, and the SARS-CoV-2 virus.

[0014] The AIE fluorescent probe is a water-soluble probe that can be dispersed in a good solvent aqueous medium to show weak fluorescence; it can aggregate in a poor solvent oil medium to show strong fluorescence. Since most biological media environments are aqueous environments, water-soluble probes are beneficial for co-incubation with viruses in biological media environments to enhance fluorescence.

[0015] The AIE fluorescent probe is prepared by sulfonating tetra-(4-hydroxyphenyl)ethylene with 1,3-propane sultone; the reaction formula is as follows:

[0016]

[0017] The molar ratio of 1,3-propane sultone to tetrakis-(4-hydroxyphenyl)ethylene is 4.5-6:1, preferably 5:1.

[0018] The sulfonation process also includes a catalyst; the catalyst includes any one of sodium ethoxide, triethylamine, trimethylamine, and pyridine; the molar ratio of the catalyst to tetra-(4-hydroxyphenyl)ethylene is 1.5 to 5:1;

[0019] The reaction medium in the sulfonation process includes one or more of ethyl acetate, ethanol, acetone, tetrahydrofuran, dimethyl sulfoxide, and dimethylformamide;

[0020] The reaction temperature during the sulfonation process is 0-60°C, the reaction environment is anhydrous and oxygen-free environment, and the reaction time is 6-18 hours. Preferably, the solution changes from dark green to orange-yellow during the reaction at a reaction temperature of 20°C, which indicates the generation of the reaction intermediate sodium salt. More preferably, the reaction temperature is 20-60°C.

[0021] The inventors have found through experiments that the yield can reach more than 50% after the sulfonation reaction time is 8 hours, and the yield does not increase significantly when the reaction time is further extended; preferably, the sulfonation reaction time is 8 to 18 hours, including 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, and 18 hours.

[0022] Furthermore, the product obtained after the sulfonation reaction is centrifuged, washed with acetone, rotary evaporated, and vacuum dried to obtain a white powdery solid.

[0023] The preparation method of TPES is very efficient, and the product yield is above 95%.

[0024] The application of the bionic heparan sulfate AIE fluorescent probe in detecting SARS-CoV-2 virus, when used, the detection method includes the steps of: mixing the AIE fluorescent probe and the SARS-CoV-2 virus in a buffer solution and then performing fluorescence detection.

[0025] The buffer is PBS buffer or artificial body fluid, which is used to simulate the pH and osmotic pressure of the physiological environment of the body.

[0026] The buffer may optionally contain Cl - 、CH3CH2COO - 、H2PO4 - 、SO4 2- 、SO3 2- 、HCO3 - Mg 2+ NH4 + One or more interfering ions in the sample, the total concentration of which is 5 mg mL -1 the following.

[0027] The AIE fluorescent probe of the present invention has extremely high stability after combining with the SARS-CoV-2 virus. Even if the mixture of the fluorescent probe and the new coronavirus is left for 30 days, the fluorescent probe does not quench over time, and the fluorescence intensity can remain constant under the interference of other ions.

[0028] The mixing time is more than 15s. The fluorescent probe binds to the virus very quickly. It only takes a short mixing time to perform fluorescence detection. The fluorescence intensity obtained by the test is stable and the test efficiency is high. It is very suitable for the simultaneous detection of large batches of samples.

[0029] The concentration of AIE fluorescent probe in the buffer solution after the AIE fluorescent probe was mixed with SARS-CoV-2 virus was 0.01 mg mL -1 Preferably, the probe concentration is 0.05 mg mL -1 The AIE fluorescent probe in the present invention is used for SARS-CoV-2 virus detection. The required probe is at the nanogram level, which saves material cost and can meet large-scale, high-demand, and wide-ranging population detection and screening.

[0030] The titer of ASARS-CoV-2 virus in the buffer solution after the AIE fluorescent probe was mixed with SARS-CoV-2 virus was 1TUmL -1 The above; The detection limit of the fluorescent probe of the present invention for the ASARS-CoV-2 virus is very low, at 1TU mL -1 The above can realize fluorescence detection.

[0031] Preferably, the titer of ASARS-CoV-2 virus is 10TU mL -1 Above; in this range, the fluorescence curve intensity is more obvious and the detection accuracy is higher. Further preferably, the titer of the ASARS-CoV-2 virus is 10~1×10 6 TU -1 .

[0032] When the Spike protein of SARS-CoV-2 virus is detected, the concentration of Spike protein in the buffer solution after the AIE fluorescent probe is mixed with SARS-CoV-2 virus is 0.001 mg mL -1 above.

[0033] The excitation wavelength of fluorescence detection is 345-370nm, the emission wavelength range is 390-600nm, and the peak fluorescence signal after the AIE fluorescent probe is mixed with the SARS-CoV-2 virus is 460-490nm.

[0034] Furthermore, the fluorescence detection conditions are fixed excitation 350nm, emission start: 370nm, emission stop: 640nm, step: 2nm, gain: 100, detection height: 7mm, and fluorescence curves after mixing different virus titers, different mutant viruses and fluorescent probes were obtained, and the peak of the fluorescence signal was at 482nm.

[0035] The AIE fluorescent probe can realize qualitative and / or quantitative detection of SARS-CoV-2 virus.

[0036] The method for quantifying SARS-CoV-2 virus using AIE fluorescent probe includes the following steps:

[0037] The initial intensity I0 of the AIE fluorescent probe buffer was tested, and the fluorescence intensity I after incubation with different titers of virus or different concentrations of Spike protein in the AIE fluorescent probe was tested. The I / I0 fluorescence enhancement degree was used as the ordinate, and the virus titer or Spike protein concentration was used as the abscissa to fit the quantitative relationship curve.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] (1) The AIE fluorescent probe in the present invention is used to detect SARS-CoV-2 virus, which is a commonly used in vitro virus detection. Compared with gene sequencing, polymerase chain reaction, and nucleic acid detection, this detection method has high sensitivity and high resolution (detection threshold is 10TU mL -1 ), low background noise, etc.

[0040] (2) The AIE fluorescent probe in the present invention is used to detect the SARS-CoV-2 virus. Nanogram-level fluorescent probes are used for one detection, and the yield of the synthesis method is above 95%, which can meet the simultaneous positive screening of a large number of people.

[0041] (3) The AIE fluorescent probe in the present invention is used to detect the SARS-CoV-2 virus. The adhesion of the fluorescent probe to the virus occurs instantly, and the accurate value of the virus titer can be obtained within 15 seconds, which can achieve a rapid detection effect, greatly reduce the detection time, and improve efficiency.

[0042] (4) The AIE fluorescent probe used in the present invention for detecting SARS-CoV-2 virus has extremely high stability. Even if the mixture of the fluorescent probe and the new coronavirus is left for 30 days, the fluorescent probe does not quench over time, and the fluorescence intensity can remain constant under the interference of other ions. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of sodium tetrakis-(4-(methoxy-1-propanesulfonate)phenyl)ethylene prepared in Example 4.

[0044] Figure 2 : Fluorescence spectra of different concentrations of TPES in Application Example 1.

[0045] Figure 3 This is the fluorescence curve of TPES dissolved in different DMSO / PBS buffers in Application Example 2.

[0046] Figure 4 This is the fluorescence curve of the mixture of TPES and the Delta variant of the new coronavirus in Application Example 3.

[0047] Figure 5 This is the fluorescence spectrum of TPES mixed with different titers of SARS-CoV-2 Omicron variants in Application Example 4.

[0048] Figure 6 Fitting quantitative curve of TPES fluorescence intensity and novel coronavirus Omicron variant titer in Application Example 4.

[0049] Figure 7 This is the fluorescence intensity of TPES mixed with different titers of SARS-CoV-2 Omicron variants in Application Example 5 for 30 days.

[0050] Figure 8 This is the fluorescence spectrum of TPES mixed with different concentrations of SARS-CoV-2 Spike protein in Application Example 6.

[0051] Fig. 9 Fitting the quantitative curve of TPES fluorescence intensity and SARS-CoV-2 Spike protein concentration in Application Example 6.

[0052] Fig.10 This is the particle size before and after the Delta variant of the new coronavirus binds to TPES in Application Example 7.

[0053] Fig.11This is the fluorescence spectrum of TPES mixed with the 2019-nCoV Omicron variant and VSV virus in Application Example 8.

[0054] Fig.12 The fluorescence intensity of the mixture of TPES and the novel coronavirus Omicron variant in Application Example 9 is affected by the interference of different ions. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Those skilled in the art can make modifications or equivalent substitutions based on the understanding of the technical scheme of the present invention without departing from the spirit and scope of the technical scheme of the present invention, and all should be included in the protection scope of the present invention.

[0056] The raw materials and viruses used in the following specific embodiments are all purchased from the market, and the solutions used in the following specific embodiments are all PBS buffer solutions unless otherwise specified.

[0057] Example 1

[0058] Add tetrakis-(4-hydroxyphenyl)ethylene (TPE-OH, 200 mg, 0.5 mmol) and sodium ethoxide (170 mg, 2.5 mmol) to a 50 ml round-bottom flask and dissolve with 10 ml of ultra-dry anhydrous ethanol. Add 0.5 ml of excess 1,3-propane sultone and react for 8 hours at 20°C, 550 rpm and argon protection. After the reaction is completed, add acetone to the reaction solution and centrifuge to remove the supernatant, repeatedly wash the precipitate, rotary evaporate and vacuum dry the organic solvent to obtain a white powdery solid, i.e., tetrakis-(4-(methoxy-1-propanesulfonic acid)phenyl)ethylene sodium TPES, with a yield of 30% and a purity of >98%.

[0059] Example 2

[0060] Add tetrakis-(4-hydroxyphenyl)ethylene (TPE-OH, 200 mg, 0.5 mmol) and sodium ethoxide (170 mg, 2.5 mmol) to a 50 ml round-bottom flask and dissolve with 10 ml of ultra-dry anhydrous ethanol. Add 0.5 ml of excess 1,3-propane sultone and react for 10 h at 30 ° C, 550 rpm, and argon protection. After the reaction is completed, add acetone to the reaction solution and centrifuge, remove the supernatant, repeatedly wash the precipitate, rotary evaporate, and vacuum dry the organic solvent to obtain a white powder solid, that is, tetrakis-(4-(methoxy-1-propanesulfonic acid)phenyl)ethylene sodium TPES, with a yield of 60% and a purity of >98%.

[0061] Example 3

[0062] Add tetrakis-(4-hydroxyphenyl)ethylene (TPE-OH, 200 mg, 0.5 mmol) and sodium ethoxide (170 mg, 2.5 mmol) to a 50 ml round-bottom flask and dissolve with 10 ml of ultra-dry anhydrous ethanol. Add 0.5 ml of excess 1,3-propane sultone and react for 14 h at 45 ° C, 550 rpm, and argon protection. After the reaction is completed, add acetone to the reaction solution and centrifuge, remove the supernatant, repeatedly wash the precipitate, rotary evaporate, and vacuum dry the organic solvent to obtain a white powder solid, that is, tetrakis-(4-(methoxy-1-propanesulfonic acid)phenyl)ethylene sodium TPES, with a yield of 70% and a purity of >98%.

[0063] Example 4

[0064] Add tetrakis-(4-hydroxyphenyl)ethylene (TPE-OH, 200 mg, 0.5 mmol) and sodium ethoxide (170 mg, 2.5 mmol) to a 50 ml round-bottom flask and dissolve with 10 ml of ultra-dry anhydrous ethanol. Add 0.5 ml of excess 1,3-propane sultone and react for 16 h at 60 ° C, 550 rpm, and argon protection. After the reaction is completed, add acetone to the reaction solution and centrifuge, remove the supernatant, repeatedly wash the precipitate, rotary evaporate, and vacuum dry the organic solvent to obtain a white powdery solid, that is, tetrakis-(4-(methoxy-1-propanesulfonic acid)phenyl)ethylene sodium TPES, with a yield of 90% and a purity of >98%.

[0065] The NMR spectrum of the product tetrakis-(4-(methoxy-1-propanesulfonic acid)benzene)ethylene sodium obtained in Example 1-4 is as follows: Figure 1 As shown, it can be seen that the target structure is obtained. According to the results of Examples 1 to 4, the TPES yields are 1<2<3<4, respectively. This is because increasing the temperature and extending the reaction time can increase the chemical reaction rate. In this regard, the inventors continued to try. When the temperature was >60°C and the reaction time was >16h, the yield did not continue to rise. Instead, the product purity decreased and the number of by-products increased. Further replacement of the reaction solvent with tetrahydrofuran, dimethyl sulfoxide, and dimethylformamide resulted in lower yields than Example 4.

[0066] Application Example 1

[0067] To study the autofluorescence emission of TPES, 10 mg of TPES was weighed and dissolved in 10 ml of PBS buffer (to simulate the virus detection environment, the solvent for subsequent experiments was PBS) to obtain 1 mg mL -1 The stock solution was diluted to obtain 0.2 mg mL -1 , 0.1mg mL -1 , 0.05mg mL -1 , 0.01mg mL -1 , 0.001mg mL -1The fluorescence emission curve of the TPES solution with a concentration gradient under 350nm excitation light was measured in a multifunctional microplate reader. The emission light range was 390-600nm. The results are shown in Figure 2 , when dissolved in PBS, the highest emission peak of TPES is at 482 nm. As the concentration of TPES increases, the fluorescence intensity increases in the entire wavelength range.

[0068] Application Example 2

[0069] To study the aggregation-induced emission effect of the AIE molecule TPES, TPES was dissolved in a gradient volume ratio of dimethyl sulfoxide (DMSO) / PBS (the volume fraction of DMSO was 20%, 40%, 60%, 80% and 100%). -1 The TPES solution was diluted 10 times with a mixed solvent of DMSO / PBS in a gradient ratio, and the fluorescence emission curves of the TPES solutions after dilution with different solvents under 350nm excitation light were measured. The test emission light range was 390-600nm, and the highest emission peak was at 482nm.

[0070] Results Figure 3 , and found that in the process of changing the PBS / DMSO solvent, as the DMSO content increased, TPES gradually began to aggregate, showing enhanced fluorescence in the full wavelength range. This shows that TPES is a water-soluble AIE, the aqueous medium is its good solvent, and DMSO is a poor solvent. Compared with the aggregation quenching of traditional fluorescent molecules, TPES exhibits an aggregation-induced emission effect.

[0071] Application Example 3

[0072] To study the detection ability of TPES for the new coronavirus, PBS was selected as the solvent to eliminate the influence of PBS. The new coronavirus Delta variant was mixed with TPES and incubated for 15 seconds, and then the fluorescence change was measured. Take 0.1 mg mL -1 TPES solution, SARS-COV-2 (B.1.617.2, Delta) was added to TPES, the volume ratio of TPES solution to Delta solution was 1:1, and the virus titer was 2×10 7 TUmL -1 , dissolved in PBS, virus and TPES were mixed for 15 seconds and then fluorescence was tested. The fluorescence curves of TPES and virus mixture, TPES, virus, and PBS were measured in an ELISA instrument. The test conditions were excitation light 350nm and emission light range 390-600nm.

[0073] Results Figure 4After TPES and the Delta variant of the new coronavirus were mixed, there was a significant fluorescence enhancement. This is because after TPES binds to the Spike protein on the surface of the virus, TPES attaches to the surface of the virus and triggers aggregation-induced emission. It can be seen that compared with TPES of the same concentration, the peak fluorescence intensity at 482nm increased by nearly 20 times, which has a very obvious change effect. In the blank control, neither PBS nor Delta had fluorescence, and only the negative control TPES had weak fluorescence in PBS. Therefore, TPES can be used to qualitatively detect whether there is a virus, and the fluorescence contrast effect is very obvious.

[0074] Application Example 4

[0075] In order to study the quantitative novel coronavirus detection capability of TPES, the virus titer gradient was experimentally determined by changing the Omicron titer.

[0076] First, dilute the SARS-COV-2 (B.1.1.529, Omicron) virus stock solution with PBS and store it in a refrigerator at a constant temperature. Take 0.1 mg mL -1 TPES solution, add 10 to 1×10 6 TU -1 Omicron of different titers (virus titers were 10TU mL -1 , 5×10 2 TU -1 , 1×10 3 TU -1 , 5×10 3 TU -1 , 1×10 4 TU -1 , 1×10 5 TU -1 , 5×10 5 TU -1 , 1×10 6 TU -1 ), the volume ratio of TPES solution to Omicron solution was 1:2, and the fluorescence test was performed after the two were mixed for 15 seconds.

[0077] To eliminate the effect of concentration on fluorescence intensity, an equal amount of PBS was added to the blank control. The fluorescence curves of different virus titers mixed with TPES and the fluorescence intensity at the emission peak of 482nm were measured in a multifunctional microplate reader. The test conditions were excitation light 350nm and emission light range 390-600nm.

[0078] Results Figure 5It was found that with the increase of virus titer, the fluorescence intensity in the entire wavelength range gradually increased. 6 TUmL -1 There are obvious differences in the detection of SARS-Cov-2, and the minimum detection threshold of TPES for SARS-Cov-2 is 1TUmL -1 , which is comparable to the detection threshold of common antibodies for detecting viruses, demonstrating the high resolution and sensitivity of fluorescence detection.

[0079] By fitting the obtained detection data, it was found that at the same fluorescent probe content, there was a quantitative relationship between the virus titer and the 482nm fluorescence intensity. The results of the fitting quantitative curve are shown in Figure 6 , the correlation curve between fluorescence enhancement degree and virus titer is: The correlation coefficient R2 = 0.99665, which is highly correlated. Therefore, for viruses of unknown titer, high-resolution TPES can be used for quantitative determination after mixing.

[0080] Application Example 5

[0081] In order to study the stability of TPES in detecting the novel coronavirus, different titers of Omicron were mixed with 0.1 mg mL -1 The volume ratio of TPES solution to Omicron solution was 1:2, and the fluorescence intensity was measured after incubation for 15 seconds. The fluorescence intensity of the TPES mixture of different titers of virus was tested after constant temperature storage for 30 days to explore the stability of the detection probe.

[0082] Results Figure 7 TPES showed no fluorescence quenching within 30 days, showing good stability. Viruses of different titers all showed good stability.

[0083] Application Example 6

[0084] Analogous to the specific binding of heparan sulfate and SARS-CoV-2, the aggregation and adhesion of TPES and the virus is the adhesion between the sulfonic acid group of TPES and the Spike protein on SARS-Cov-2. In order to prove the adhesion between the two, the experiment incubated TPES and the new coronavirus Spike protein and observed the fluorescence changes.

[0085] TPES was used in the detection experiment of the new coronavirus Spike protein. Different concentrations of SARS-CoV-2 Spike Protein (RBD, mFc Tag) were prepared in PBS, including 0.5 mg mL -1 , 0.1mg mL -1 , 0.05mgmL-1 , 0.01mg mL -1 , 0.005mg mL -1 , 0.001mg mL -1 , 0.0005mg mL -1 , 0.0001mg mL -1 , stored at a constant temperature in the refrigerator.

[0086] Take 0.03 mg mL -1 TPES solution was added to TPES, and the Spike protein solution was mixed evenly. The volume ratio of TPES solution to Spike protein solution was 1:2. The fluorescence test was performed after the two were mixed for 15 seconds. To eliminate the effect of concentration on fluorescence intensity, an equal amount of PBS was added to the blank control. The fluorescence curves after mixing different virus titers with TPES and the fluorescence intensity at the emission peak of 482nm were measured respectively. The test conditions were excitation light 350nm and emission light range 390-600nm.

[0087] Results Figure 8 After incubation, there was an obvious fluorescence enhancement. There was a quantitative relationship between the concentration of Spike protein and the fluorescence intensity at 482 nm. The quantitative relationship curve is shown in Fig. 9 , the correlation curve between fluorescence enhancement degree and Spike protein concentration is: The correlation coefficient of the fitting curve is R2=0.9992, which proves that the binding of TPES and virus is based on the adhesion of TPES to the Spike protein.

[0088] Application Example 7

[0089] To further study the principle of TPES in detecting the novel coronavirus, the particle size change before and after virus adhesion was tested. -1 TPES solution, SARS-COV-2 (B.1.617.2, Delta) was added to TPES, the volume ratio of TPES solution to Delta solution was 1:1, and the virus titer was 2×10 7 TUmL -1 , dissolved in PBS. The new coronavirus Delta variant was mixed with TPES and incubated for 15 seconds before measuring the change in particle size.

[0090] Results Fig.10 Delta combined with TPES, and the particle size changed from 177nm to 198nm. This is because TPES is enriched on the surface of the viral Spike protein.

[0091] Application Example 8

[0092] In order to prove that TPES aggregates after binding to the Spike protein of SARA-CoV-2, TPES and VSV vesicular stomatitis virus were subjected to the same experimental verification. VSV virus is an RNA virus with VSV protein outside its envelope.

[0093] In the virus detection specificity test experiment of TPES, 0.02 mg mL -1 TPES and Pseudovirus-SARS-COV-2 (B.1.1.529, Omicron) and Pseudovirus-VSV, both viruses were dissolved in PBS, and the virus titer was 1×10 7 TUmL -1 , the volume ratio of TPES solution to VSV solution is 1:1. The fluorescence curve of TPES probe was measured, and the test conditions were consistent with the above. To eliminate the influence of concentration on fluorescence intensity, an equal amount of PBS was added to the blank control. The fluorescence curves of different virus titers mixed with TPES and the fluorescence intensity at the emission peak of 482nm were measured respectively. The test conditions were excitation light 350nm and emission light range 390-600nm.

[0094] Results Fig.11 At the same virus titer, VSV virus cannot cause aggregation-induced luminescence of TPES, and the fluorescence curve is basically overlapped with that of TPES PBS solution.

[0095] Application Example 9

[0096] In order to study whether the novel coronavirus detection ability of TPES would be interfered by other ions, the detection fluctuations under the interference of multiple ions were further measured.

[0097] In the virus detection ion interference test experiment of TPES, at 0.1 mg mL -1 TPES and 1×10 5 TUmL -1 Add 1 mg mL -1 The volume ratio of TPES solution and Omicron solution is 1:1. Ions include: Cl - 、CH3CH2COO - 、H2PO4 - 、SO4 2- 、SO3 2- 、HCO3 - Mg 2+ NH4 +TPES, Omicron, and ions were dissolved in PBS. To eliminate the effect of concentration on fluorescence intensity, an equal amount of PBS was added to the blank control. Blank refers to 0.1 mg mL -1 TPES and 1×10 5 TUmL -1 The Omicron mixture was used to measure the fluorescence intensity at the 482nm emission peak under different ion interferences. The test conditions were excitation light 350nm and emission light range 390-600nm.

[0098] Results Fig.12 , it can be seen that even in Cl - 、CH3CH2COO - 、H2PO4 - 、SO4 2- 、SO3 2- 、HCO3 - Mg 2+ NH4 + In the presence of , there is almost no obvious change in the fluorescence intensity after TPES and the new coronavirus combine, indicating that the detection method has strong anti-interference ability.

Claims

1. Application of a bionic heparan sulfate AIE fluorescent probe in detecting SARS-CoV-2 virus for non-disease diagnosis or treatment purposes, characterized in that: The structural formula of the AIE fluorescent probe is as follows:

2. The use of the bionic heparan sulfate AIE fluorescent probe according to claim 1 in detecting SARS-CoV-2 virus for non-disease diagnosis or treatment purposes, characterized in that: The SARS-CoV-2 virus is any one or more of the variant Omicron, the variant Delta and the wild species.

3. The use of the bionic heparan sulfate AIE fluorescent probe according to claim 1 in detecting SARS-CoV-2 virus for non-disease diagnosis or treatment purposes, characterized in that: The AIE fluorescent probe can realize qualitative and / or quantitative detection of SARS-CoV-2 virus.

4. The use of the bionic heparan sulfate AIE fluorescent probe according to claim 1 or 3 in detecting SARS-CoV-2 virus for non-disease diagnosis or treatment purposes, characterized in that: The method for quantifying SARS-CoV-2 virus using AIE fluorescent probe includes the following steps: The initial intensity I0 of the AIE fluorescent probe buffer was tested, and the fluorescence intensity I after incubation with different titers of virus or different concentrations of Spike protein in the AIE fluorescent probe was tested. The I / I0 fluorescence enhancement degree was used as the ordinate, and the virus titer or Spike protein concentration was used as the abscissa to fit the quantitative relationship curve.

5. The use of the bionic heparan sulfate AIE fluorescent probe according to claim 1 in detecting SARS-CoV-2 virus for non-disease diagnosis or treatment purposes, characterized in that: The application includes the following steps: mixing the AIE fluorescent probe with the SARS-CoV-2 virus in a buffer solution and then performing fluorescence detection.

6. The use of the bionic heparan sulfate AIE fluorescent probe according to claim 5 in detecting SARS-CoV-2 virus for non-disease diagnosis or treatment purposes, characterized in that: The buffer is PBS buffer; And / or, the mixing time is more than 15 seconds.

7. The use of the bionic heparan sulfate AIE fluorescent probe according to claim 5 in detecting SARS-CoV-2 virus for non-disease diagnosis or treatment purposes, characterized in that: The concentration of AIE fluorescent probe in the buffer solution after the AIE fluorescent probe was mixed with SARS-CoV-2 virus was 0.01 mg mL -1 above; And / or, the titer of ASARS-CoV-2 virus in the buffer after the AIE fluorescent probe is mixed with SARS-CoV-2 virus is 1TU mL -1 above; When the Spike protein of SARS-CoV-2 virus is detected, the concentration of Spike protein in the buffer solution after the AIE fluorescent probe is mixed with SARS-CoV-2 virus is 0.001 mg mL -1 above.

8. The use of the bionic heparan sulfate AIE fluorescent probe according to claim 5 in detecting SARS-CoV-2 virus for non-disease diagnosis or treatment purposes, characterized in that: The excitation wavelength of fluorescence detection is 345-370nm, the emission wavelength range is 390-600nm, and the peak fluorescence signal after the AIE fluorescent probe is mixed with the SARS-CoV-2 virus is 460-490nm.

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