Method for detecting and inactivating arbovirus

Abstract

A detection and inactivation integrated method of arbovirus, comprising the following steps: mixing and treating biotin-modified G-rich DNA single strand with hematin to obtain DNA enzyme with catalytic effect; simultaneously, forming double-labeled nanoballs by double labeling streptavidin and anti-virus antibody on the surface of nanoballs with carboxyl; incubating the sample to be tested with anti-virus monoclonal antibody, double-labeled nanoballs and biotinylated DNA enzyme in sequence, separating and washing to obtain immune complexes; adding H2O2 which can be catalytically hydrolyzed by DNA enzyme and fluorescent red molecules which can occur oxidation reaction to the immune complexes to obtain oxidation products with AIE property; judging whether the sample to be tested contains the virus to be tested by the significant change of AIE molecule fluorescence from nothing to something; the AIE molecule formed by the oxidation products has photodynamic activity, generates reactive oxygen species under white light radiation, and acts on the virus in situ to realize the inactivation of the virus.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and in particular relates to an integrated method for detecting and inactivating arboviruses. Background Technology

[0002] Vector-borne viruses (such as Zika virus, dengue virus, and chikungunya virus, which are transmitted to humans via mosquitoes) account for more than 17% of all infectious diseases, causing hundreds of millions of infections and millions of deaths annually, posing a serious threat to public health. For example, Zika virus spreads and circulates among humans via Aedes mosquitoes. Zika virus infection during pregnancy can lead to microcephaly and other congenital malformations in infants, or premature birth and miscarriage. In February 2016, the WHO declared Zika virus-associated microcephaly a Public Health Emergency of International Concern. Dengue virus is transmitted among humans by Aedes albopictus and Aedes aegypti mosquitoes. This mosquito-borne viral infection is dengue fever, which is more common in tropical and subtropical climates. Worldwide, there are approximately 50 to 100 million dengue fever infections and about 20,000 deaths. The spread of the chikungunya virus can cause chikungunya fever. Patients bitten by infected mosquitoes will suddenly develop fever, headache, nausea, fatigue, and often severe joint pain. The symptoms usually last for a few days, but can last for weeks, months or even years.

[0003] Numerous studies and clinical practices have demonstrated that early detection, early diagnosis, and early treatment are crucial for preventing infectious diseases and reducing mortality rates. However, while traditional enzyme-linked immunosorbent assays (ELISA) are convenient, their low sensitivity stems from using single-molecule enzymes as signal amplification units. Quantitative real-time PCR (qPCR) offers high sensitivity and specificity, but requires expensive equipment and skilled personnel, limiting its widespread application in resource-poor regions. Furthermore, the aerosols generated during PCR amplification can pollute the environment, leading to false positives. In recent years, various sensitive virus analysis methods have emerged, such as surface-enhanced Raman spectroscopy, lateral flow immunochromatography, self-enrichment electrospinning, label-free plasmon resonance spectroscopy, and fluorescence analysis. Aggregation-induced emission (AIE)-based fluorescence analysis has garnered significant attention due to its high sensitivity, ease of operation, and lack of complex equipment requirements. However, current research primarily focuses on virus detection, neglecting the secondary pollution and reinfection of the environment caused by live viruses present in waste samples. Developing an integrated method for virus detection and inactivation to prevent reinfection in the population is a pressing issue.

[0004] Currently, vaccines are considered one of the many effective methods to prevent viral infection; however, obtaining vaccines is often time-consuming, requires specialized equipment, and necessitates significant financial and material resources. While some new methods have been applied to inactivate viruses—such as heat, ultraviolet light, chlorine-based disinfectants, and nano-silver—these methods have been proven to inactivate viruses and block their transmission. However, these methods also carry some other uncertain side effects.

[0005] Therefore, the research objective of this invention is to obtain a highly sensitive and efficient method for detecting and inactivating arboviruses without significant side effects. Summary of the Invention

[0006] The purpose of this invention is to provide a method for detecting and inactivating vector-borne viruses, aiming to solve the technical problems of existing virus detection methods such as low sensitivity, complex detection operation, susceptibility to external interference, and poor detection accuracy. At the same time, it adds a virus inactivation module to the existing virus detection method to realize a new technology that integrates virus detection and inactivation, effectively preventing secondary pollution and spread of viruses to the environment.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An integrated method for detecting and inactivating arboviruses, comprising the following steps: Step 1: React biotinylated single-stranded DNA with heme to obtain biotin-labeled DNA enzyme; Step 2: Modify the carboxyl-modified nanospheres with polyclonal antibodies against the target virus and streptavidin molecules to obtain dual-labeled nanospheres; Step 3: Incubate the sample to be tested with the monoclonal antibody against the virus to be tested for the first time, then separate and wash to obtain the first complex; incubate the first complex with the double-labeled nanospheres for the second time, then separate and wash to obtain the second complex; add biotin-labeled DNase to the second complex for the third incubation, then separate and wash to obtain the third complex; add fluorescent red and hydrogen peroxide to the third complex for the fourth incubation. If significant fluorescence is present, the sample to be tested is determined to contain the virus to be tested. Step four: Irradiate the incubated system under a light source to inactivate the virus to be tested.

[0008] The relevant content in the above technical solution is explained as follows: 1. In the above scheme, the virus to be tested is one of Zika virus, dengue virus, and chikungunya virus; If the arbovirus to be tested is Zika virus, the monoclonal antibody against the virus to be tested is an antibody against Zika virus gE protein, and the polyclonal antibody against the virus to be tested is an antibody against Zika gE protein. For example, when the arbovirus to be tested is dengue virus, the monoclonal antibody against the virus to be tested is a dengue virus gE antibody, and the polyclonal antibody against the virus to be tested is a dengue virus gE antibody. For example, when the arbovirus to be tested is Chikungunya virus, the monoclonal antibody against the virus to be tested is Chikungunya virus E1 antibody, and the polyclonal antibody against the virus to be tested is Chikungunya virus E1 protein antibody.

[0009] 2. In the above scheme, the biotinylated single-stranded DNA is a nucleic acid modified with biotin and rich in the base G, and the biotin-labeled DNase has peroxidase catalytic activity. The specific preparation steps for obtaining heme / G-quadruplex DNase (G4 / hemin DNase) from single-stranded DNA and heme are existing technologies. The selection of which single-stranded DNA sequence to use should be known to those skilled in the art, and therefore will not be elaborated here. Specifically, the single-stranded DNA in this technical solution is biotinylated single-stranded DNA.

[0010] 3. In the above scheme, in step one, the biotinylated single-stranded DNA is first heated to 65~90℃, the temperature range for reacting with heme is 20~30℃, and the pH range for the reaction is 7.0~7.8.

[0011] 4. In the above scheme, in step two, nanospheres with a particle size of 200-800 nm are selected. The specific modification steps are as follows: Nanospheres containing carboxyl-modified groups, polyclonal antibodies against the target virus, and streptavidin are incubated at a temperature of 20-37°C for at least 4 hours to obtain dual-labeled nanospheres simultaneously labeled with polyclonal antibodies against the target virus and streptavidin. The specific modification principle is that two or more proteins are modified onto the surface of the nanospheres through a covalent coupling reaction between the carboxyl groups on the nanosphere surface and the amino groups of the proteins.

[0012] 5. In the above scheme, in step two, the mass ratio of the polyclonal antibody against the virus to be tested and streptavidin is in the range of 0.5:1 to 6:1.

[0013] 6. In the above scheme, in step three, the sample to be tested is co-incubated with the enzyme-linked immunosorbent assay (ELISA) plate containing the monoclonal antibody against the virus to be tested at a temperature of 37°C for more than 2 hours. After washing the ELISA plate, it is blocked and washed again to obtain the first complex.

[0014] 7. In the above scheme, in step three, the temperature of the second incubation is 25~37℃ and the incubation time is greater than 1 hour; the temperature of the third incubation is 25~37℃ and the incubation time is greater than 0.5 hours; the temperature of the fourth incubation is 20~30℃ and the incubation time is greater than 0.5 hours.

[0015] 8. In the above scheme, the concentration range of fluorescent red molecules is 1 μM to 60 μM, and the concentration range of hydrogen peroxide is 0.1 mM to 6 mM.

[0016] 9. In the above scheme, this technical solution can not only perform qualitative detection but also quantitative detection of arboviruses. In step three, fluorescence detection at a wavelength of 590 nm is used to quantitatively detect the virus to be tested. The experimental steps for quantitative detection are basic methods known to those skilled in the art and will not be elaborated here.

[0017] 10. In the above scheme, the light source is white light, and the irradiation time is 1 to 60 minutes.

[0018] 11. In the above scheme, in step two, a secondary antibody against the target virus can be first modified onto the carboxyl-modified nanospheres, followed by modification with a polyclonal antibody against the target virus and streptavidin molecules to obtain dual-labeled nanospheres. However, modifying the secondary antibody does not... It is necessary, but only to save costs.

[0019] The working principle of this invention is: This invention provides an integrated analytical method for virus detection and inactivation. By constructing a DNA-based biomimetic enzyme system mediated by dual-labeled nanospheres, signal amplification can be effectively achieved. Streptavidin on the surface of the nanospheres binds to biotinylated DNase; multiple streptavidin molecules can be bound to the surface of one nanosphere, and one streptavidin molecule can bind to four biotin molecules. This allows multiple enzyme molecules to be bound to the immunosensor, improving the sensitivity of the immunoassay method. Simultaneously, fluorescent red and hydrogen peroxide are added as substrates for the catalytic reaction. Hydrogen peroxide is catalyzed and hydrolyzed by the DNase, further oxidizing the fluorescent red molecules to produce AIE molecules, which emit bright fluorescence in the detection solution, thus achieving the detection of the target virus. At the same time, the AIE molecule is a photosensitizer that can generate ROS, acting on the virus in situ, thereby achieving highly efficient virus inactivation. This method uses biomimetic enzymes as signal amplification units, which can greatly improve the stability and reduce the cost of the method compared with the use of natural enzymes. Using dual-labeled nanospheres as bridging agents can couple multiple DNA enzyme molecules, which can greatly improve the sensitivity of the analytical method compared with the single-molecule enzyme in the traditional enzyme-linked immunosorbent assay. More importantly, this invention incorporates photodynamic inactivation of viruses into the immunoassay method, which effectively solves the problem of secondary pollution of the environment caused by live viruses in waste, as well as prevents the re-spread of viruses and the spread of diseases.

[0020] Due to the application of the above technical solution, the present invention has the following advantages and effects compared with the prior art: 1. This invention utilizes dual-labeled nanospheres as a bridge, which can provide multiple sites for virus recognition and binding to biotinylated DNAase, thus significantly improving the sensitivity of the detection method.

[0021] 2. This invention uses DNase as a signal amplification source, which can significantly reduce the overall detection cost and improve the stability of the detection method compared with the currently mature methods that use natural enzymes.

[0022] 3. This invention combines virus detection and virus eradication into one, which can greatly reduce the risk of infection of detection operators compared with single-mode virus detection, while blocking the risk of secondary transmission of the virus.

[0023] This technical solution provides a novel photodynamic therapy modality for disease treatment. It utilizes photosensitizers to generate reactive oxygen species under light radiation, causing irreversible damage to cellular proteins and nucleic acids. Therefore, this approach is of great significance for preventing viral diseases and controlling the spread of viruses. Attached Figure Description

[0024] Figure 1 This is a schematic diagram illustrating the principle of the integrated virus detection and inactivation method provided in this embodiment of the invention; Figure 2 These are the experimental results of the catalytic oxidation activity determination of biotinylated DNAase and the photodynamic properties of the oxidation product AIE molecule in the embodiments of the present invention; Figure 3 Chromatographic and high-resolution mass spectrometry results of oxidation products; Figure 4 The results of the characterization of the dual-labeled nanospheres and the fluorescence immunoassay method used for Zika virus specific detection in the embodiments of the present invention are shown. Figure 5 The figure shows the quantitative detection results of the Zika virus detection method provided in the embodiments of the present invention. Figure 6 This is a diagram showing the dengue virus-specific detection results in an embodiment of the present invention; Figure 7 This is a diagram showing the specific detection results of Chikungunya virus in an embodiment of the present invention; Figure 8 This is a graph showing the detection results of Zika virus in clinical samples in an embodiment of the present invention; Figure 9 The effect of oxidized fluorescent red on host Vero cells under white light and dark conditions in this embodiment of the invention; Figure 10 This is a graph showing the verification results of using the oxidation product as a photosensitizer for Zika virus inactivation in the embodiments of the present invention. Figure 11This is a graph showing the verification results of using the oxidation product as a photosensitizer for dengue virus inactivation in the embodiments of the present invention; Figure 12 This is a diagram showing the verification results of using the oxidation product as a photosensitizer for the inactivation of Chikungunya virus in an embodiment of the present invention. Implementation

[0025] To make the objectives, technical solutions, and technical effects of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. In conjunction with the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] In the description of this invention, it should be understood that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "multiple" means two or more, unless otherwise explicitly specified.

[0027] The weights of the relevant components mentioned in the embodiments of this invention can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this invention is within the scope disclosed in the embodiments of this invention. Specifically, the weights mentioned in the embodiments of this invention can be well-known units of mass in the chemical industry, such as μg, mg, g, and kg.

[0028] This invention provides a method for detecting and inactivating viruses, comprising the following steps: S1. Obtain a DNA enzyme with horseradish peroxidase catalytic properties; S2. Obtain double-labeled nanospheres; S3. The sample to be tested is incubated with the monoclonal antibody against the virus to be tested for the first time, separated and washed to obtain the first complex; S4. Add the double-labeled nanospheres to the first composite for a second incubation, then separate and wash to obtain the second composite; S5. Add biotin-labeled DNase to the second complex for a third incubation, separate and wash to obtain the third complex; S6. Fluorescent red and hydrogen peroxide molecules are added to the third complex for a fourth incubation. The oxidation product can significantly produce fluorescence, which can be used to determine whether the sample to be tested contains a virus. S7. Irradiate the above oxidation product with white light to determine whether the oxidation product, as a photosensitizer, can inactivate the virus.

[0029] The method for detecting and inactivating viruses provided in this embodiment of the invention, Figure 1 This is a schematic diagram illustrating the virus detection principle provided in an embodiment of the present invention. A horseradish peroxidase-based DNase is constructed as a signal amplification source. Simultaneously, dual-labeled nanospheres are introduced to simultaneously recognize the target virus and efficiently bind to the DNase. Multiple DNase molecules are introduced into the immune complex, further amplifying the signal. Enzymatic hydrolysis of hydrogen peroxide oxidizes fluorescent red molecules to generate AIE molecules, thus producing fluorescence and enabling the detection of the target virus. Furthermore, the obtained AIE molecules possess photodynamic activity; upon irradiation with white light, they generate reactive oxygen species that act on the virus in situ, thereby inactivating the virus. Ultimately, this achieves an integrated method for virus detection and inactivation. When the target virus is present, the antiviral monoclonal antibody bound to the ELISA plate can specifically capture the target virus. Then, the dual-labeled nanospheres, through the antiviral polyclonal antibody bound to their surface, can recognize the target virus, forming an immunocomplex sandwich. A synthesized biotin-labeled DNase is added to the immunocomplex sandwich. The streptavidin coupled to the nanosphere binds to the biotin-labeled DNase in the immunocomplex, further catalyzing the hydrolysis of added hydrogen peroxide and oxidizing the added fluorescent red molecules, emitting a bright fluorescence, thereby realizing the detection of the virus. At the same time, the oxidation product has photodynamic properties and can inactivate the virus in situ under white light irradiation. When the target virus is not present, the virus cannot be captured and is eluted, and the subsequent immune reaction cannot proceed. Therefore, the added fluorescent red molecules cannot be catalyzed and the solution is fluorescenceless. The detection of the target virus is achieved by the fluorescence in the solution.

[0030] Preferably, in step S01 above, biotin-labeled single-stranded DNA is reacted with heme at an appropriate molar concentration to examine the catalytic oxidation activity of the synthesized DNase and optimize the optimal concentrations of DNase, hydrogen peroxide, and fluorescent red; at the same time, the photodynamic properties of the oxidation product are examined.

[0031] Preferably, in step S02 above, the preparation method of dual-labeled nanospheres is as follows: First, carboxyl-containing nanospheres are prepared. 100 μL of nanospheres (10 mg / mL) are activated for 30 min in 500 μL of phosphate buffer (10 mM, pH=6.8) with 4 mg / mL EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 2 mg / mL NHS (N-hydroxysuccinimide). The activated nanospheres are centrifuged, washed three times with phosphate buffer (10 mM, pH=7.2), and then resuspended in 455 μL of phosphate buffer (10 mM, pH=7.2). Then, 30 μL of streptavidin (1 mg / mL) and 8 μL of 1.18 mg / mL polyclonal antibody against the target virus are added to the activated nanospheres and incubated at 37°C for 4–10 hours. Finally, the prepared dual-labeled nanospheres were centrifuged and washed three times to achieve a final concentration of 1 mg / mL. They were then stored in a phosphate buffer of 1% bovine serum albumin at 4°C for later use.

[0032] Preferably, in step S02 above, if the virus to be tested is Zika virus, the added polyclonal antibody is an anti-Zika virus gE protein antibody; if the virus to be tested is dengue virus, the added polyclonal antibody is an anti-dengue virus gE protein antibody; if the virus to be tested is chikungunya virus, the added polyclonal antibody is an anti-chikungunya virus E1 protein antibody.

[0033] In the virus detection and eradication method provided in this invention, if the detected virus is Zika virus, the monoclonal antibody is an anti-Zika virus gE protein monoclonal antibody, and the polyclonal antibody is an anti-Zika virus gE protein antibody; if the detected virus is dengue virus, the monoclonal antibody is an anti-dengue virus gE monoclonal antibody, and the polyclonal antibody is an anti-dengue virus gE protein antibody; if the detected virus is chikungunya virus, the monoclonal antibody is an anti-chikungunya virus E1 protein antibody, and the polyclonal antibody is an anti-chikungunya virus E1 protein antibody. Simultaneously, the virus detection result is determined by detecting the fluorescence intensity at 590 nm; the inactivation effect of the virus is determined by the product after oxidation under white light through plaque assays and host cell activity tests.

[0034] This invention has undergone multiple experiments. Some of the experimental results are presented first as a reference for further detailed description of the invention. The following is a detailed description in conjunction with specific embodiments.

[0035] The prepared biotinylated DNA enzyme (biotin-5'-AAAAAGGGTAGGGCGGGTTGGG-3) was reacted with hydrogen peroxide and fluorescent red. During the reaction period of 0–60 min, the fluorescence peak of the oxidation product was clearly detected, with the maximum emission peak corresponding to a wavelength of 590 nm. Figure 2a) A mixture of control single-stranded DNA (biotin-5'-TATAGTTGACAGAGACTTGTTA-3') incubated with heme was further incubated with hydrogen peroxide and fluorescein. No significant fluorescence peak was observed after the same reaction time, and other control groups also showed no obvious fluorescence peaks. The experimental results indicate that the prepared biotinylated DNAase can catalyze the oxidation of fluorescein molecules with hydrogen peroxide. Further testing of the reaction kinetics revealed that the reaction reached its maximum fluorescence intensity within 30 minutes, and the red fluorescence emitted by the oxidation product, halogen, was clearly visible under 470 nm blue light. This also demonstrated that the oxidation product, halogen, possesses AIE properties; it exhibits no fluorescence in dimethyl sulfoxide, but the fluorescence gradually increases with increasing water content, reaching an 1100-fold increase when the water volume fraction is 90%.

[0036] To optimize the fluorescence intensity of the oxidation product at 590 nm, the concentrations of DNase, hydrogen peroxide, and fluorescent red were further optimized. It was found that as the DNase concentration increased, the fluorescence of the oxidation product gradually increased, eventually reaching saturation. Figure 2 c), the final optimized concentrations of DNase, hydrogen peroxide, and fluorescent red were 200 nM, 1 mM, and 20 μM, respectively. Subsequent experiments were conducted under these optimized concentration conditions. Under these optimal concentration conditions, it was found that the absorption in the 400–620 nm range gradually increased with increasing reaction time. This broad absorption range allows the oxidation product to utilize its photodynamic activity for virus inactivation under white light irradiation. Furthermore, UV-Vis spectrophotometry showed that the peak shape and position of the oxidation product were completely consistent with those of pure halogen, indicating that the oxidation product was indeed halogen (…). Figure 2 d). The results of chromatography and mass spectrometry further confirmed that the oxidation product was halogenated ( ). Figure 3 ).

[0037] To further verify that the oxidation products can generate reactive oxygen species under light conditions, solutions of "DNase + hydrogen peroxide + fluorescein", fluorescein, hydrogen peroxide, halogenated fluorescein, dihydroporphyrin, and rose red were incubated with dichlorofluorescein, as well as solutions of "DNase + hydrogen peroxide + fluorescein", dichlorofluorescein, fluorescein, dihydroporphyrin, and rose red alone. These solutions were irradiated with white light for up to 20 minutes, and the fluorescence intensity at 525 nm was examined. Dihydroporphyrin and rose red were incubated with dichlorofluorescein as positive control groups. The experimental results showed that after incubation of "DNase + hydrogen peroxide + fluorescein" and halogenated fluorescein with dichlorofluorescein, obvious fluorescence peaks appeared. Compared with the control groups, the fluorescence intensity at 590 nm increased by 655 times and 537 times, respectively. Figure 2(e, f) indicates that the oxidation product of biotinylated DNAase catalyzing the hydrolysis of hydrogen peroxide and oxidation of fluorescent red molecules has significant photodynamic activity and can generate reactive oxygen species.

[0038] To verify the types of reactive oxygen species generated, commercially available probes for monitoring singlet oxygen generation, 9,10-anthratridimyl-bis(methylene)dicarboxylic acid (ABDA), hydroxyl radical indicator probe hydroxyphenylfluorescein (HPF), and superoxide ion indicator probe dihydrorhodamine 123 (DHR), were incubated with "DNase + hydrogen peroxide + fluorescent red" and halogenated, respectively. The results showed that both "DNase + hydrogen peroxide + fluorescent red" and halogenated could generate singlet oxygen (…). Figure 2 g), hydroxyl radicals ( Figure 2 h) and superoxide ions (h) Figure 2 i), which provides a solid theoretical basis for the subsequent inactivation of the virus.

[0039] The hydration size and zeta potential of the dual-labeled and unlabeled nanospheres were tested. The results showed that the average hydration size of the nanospheres labeled with the antibody and streptavidin increased from 361.3 nm to 407.9 nm. Figure 4 a), the average Zeta potential decreased from -38.2 mV to -50.7 mV ( Figure 4 (b) This indicates that the two proteins were successfully modified on the surface of the nanospheres. The dual-labeled nanospheres were further incubated with different concentrations of target virus. After centrifugation and washing, nucleic acid was extracted and analyzed by quantitative real-time PCR. The PCR analysis results showed that as the virus concentration increased, the cycle number gradually decreased, and the peak shape of the result curve was completely consistent with the positive result (VIII), while the unmodified nanospheres showed a negative result (b). Figure 4 c). These results indicate that the dual-labeled nanospheres can successfully recognize the target virus.

[0040] In step S03, 100 μL of 4 ng / μL monoclonal anti-Zika E protein antibody was added to an enzyme-linked immunosorbent assay (ELISA) plate and incubated overnight at 4°C. The plate was then washed three times with PBST (1×PBS buffer containing 0.05% Tween 20, pH 7.4), followed by incubation for 2 hours at room temperature with blocking buffer (2% fetal bovine serum albumin). The plate was then washed twice with PBST. Different concentrations of Zika virus were added to the plate and incubated at 37°C for 1 hour, followed by washing four times with PBST. 60 μL of double-labeled globulins (1 mg / mL) were added to the immune complex and incubated at 37°C for 1 hour. The plate was then washed four times with PBST, followed by adding 100 μL of B-G4DH (200 nM) and incubating at 37°C for 1 hour. The plate was then washed four times with PBST. Next, add 100 μL of HEPES buffer (20 mM HEPES, 10 mM KCl, 100 mM NaCl, pH=7.3) containing 1 mM hydrogen peroxide and 20 μM fluorescent red to the ELISA plate, and continue incubation at room temperature for 30 minutes. Collect the test solution for fluorescence spectroscopy determination; the presence of the target virus in the solution is determined by the fluorescence results.

[0041] This invention includes a control group consisting of other arboviruses, namely dengue 1, dengue 2, dengue 3, dengue 4, chikungunya virus, Japanese encephalitis virus, and a phosphate-buffered saline (PBRS) reagent blank. All other experiments are identical to those described above.

[0042] The detection results of this embodiment are as follows: Figure 4 As shown in d, fluorescence was only detected in the target Zika virus detection solution, and the solution showed significant red fluorescence under 470nm blue light illumination. In contrast, no significant red fluorescence was observed in the other negative and blank control groups, and the corresponding fluorescence signal could not be collected by the fluorescence spectrometer. Figure 4 d). The experimental results demonstrate the high specificity of this method.

[0043] Figure 5 To apply this method to the quantitative detection of Zika virus, both one-step and two-step methods were employed. The one-step method involved mixing biotinylated DNase with dual-labeled nanospheres, followed by incubation with the captured target virus. Experimental results showed that fluorescence gradually increased with increasing target Zika virus concentration, and the virus concentration range was within 4 × 10⁻⁶. 3 to 2 × 10 5 The results showed a good linear relationship with PFU / mL, and the limit of detection for the one-step method was calculated to be 3.37 × 10⁻⁶. 3PFU / mL. The two-step method is performed according to S3~S5 as described above. The results show that as the virus concentration increases, the fluorescence of the detection solution gradually increases, and the virus concentration range is 1 × 10⁻⁶. 2 to 1 × 10 4 The PFU / mL ratio showed a good linear relationship, with a detection limit of 66.3 PFU / mL. The detection limit of the one-step method was significantly higher than that of the two-step method. This is mainly because the biotin-modified DNase binds first to the double-labeled nanospheres, and steric hindrance may affect the binding of the recognition antibody on the double-labeled nanospheres to the captured virus.

[0044] By changing the antiviral monoclonal antibody and the corresponding antiviral polyclonal antibody bound to the ELISA plate, specific detection of viruses such as dengue virus and chikungunya virus can be easily achieved. Other experimental procedures are completely consistent with those described above. When the virus to be detected is dengue virus, Zika virus, chikungunya virus, Japanese encephalitis virus, and reagent blank are used as control groups. The results are as follows: Figure 6 As shown; when the virus to be tested is Chikungunya virus, Zika virus, dengue virus, Japanese encephalitis virus, and reagent blank are used as control groups, and the results are as follows. Figure 7 As shown.

[0045] Twenty urine samples and twenty serum samples were obtained from healthy volunteers. These biological samples were diluted tenfold with phosphate buffer, and then different concentrations of virus were added to the diluted samples. The detection of Zika virus in complex samples was achieved using the detection systems constructed in Examples 1 and 2. Figure 8 As shown, 14 out of 20 urine samples and 15 out of 20 blood samples were positive. Different viral concentrations in the samples could also be distinguished by the fluorescence intensity of the detection solution at 590 nm. These results were completely consistent with the gold standard quantitative PCR results. This demonstrates that the invention effectively avoids interference from complex systems, possesses strong anti-interference capabilities, and has high accuracy, enabling the detection of clinical samples.

[0046] In step S7 above, to verify whether oxidized fluorescent red can inactivate the virus under white light irradiation, it is first necessary to verify whether oxidized fluorescent red affects the cell viability of host cells under optimized concentration conditions. The "DNase + hydrogen peroxide + fluorescent red" mixture was divided into two groups: one group was irradiated with white light for 1 hour, and the other group was placed in the dark for 1 hour. The cells were then incubated in a 37°C CO2 incubator for 24, 48, and 72 hours, respectively. The cell supernatant was discarded, and 100 μL of thiazolyl blue (final concentration 0.5 mg / mL) was added. The cells were incubated in a 37°C CO2 incubator for 4 hours. The supernatant was then aspirated, and 100 μL of dimethyl sulfoxide was added to each well. The absorbance at 570 nm was measured using a microplate reader. The experimental results are as follows: Figure 9 As shown, when Vero cells were incubated with "DNase + hydrogen peroxide + fluorescent red", the cell viability remained above 90% for 72 hours under both light and non-light conditions, indicating that the effect of the current optimized experiment on Vero cells alone is negligible.

[0047] Further virus inactivation experiments were conducted under the optimized conditions described above. The experiments were divided into 6 groups, including: (I) Vero cell control group; (II) Target virus + 200 nM biotinylated DNase + 1 mM hydrogen peroxide + 20 μM fluorescent red / white light irradiation for 1 hour; (III) Target virus + 200 nM biotinylated DNase + 1 mM hydrogen peroxide + 20 μM fluorescent red / dark conditions for 1 hour; (IV) Target virus / white light irradiation for 1 hour; (V) Target virus / dark conditions for 1 hour; (VI) Target virus was not treated in any way, but was treated according to the above 6 experimental groups, and then added to Vero cells for incubation for 1 hour. The supernatant was then removed, and maintenance medium containing low melting point agarose gel was added for continued culture for different number of days. The cells were then fixed with 4% paraformaldehyde for 2 hours, and the gel was stained with 0.5% crystal violet. Empty plaques were observed, and the number of blanks and virus titer were calculated. Figure 10 As can be seen, the group irradiated with the target virus + 200nM biotinylated DNase + 1mM hydrogen peroxide + 20μM fluorescent red / white light for 1 hour showed no empty spots, which was completely consistent with the Vero control group. However, other groups clearly showed blank areas. This indicates that under white light irradiation, the combination of "200nM biotinylated DNase + 1mM hydrogen peroxide + 20μM fluorescent red" produces reactive oxygen species, which inactivate the virus, preventing it from infecting host cells and replicating progeny viruses. Statistical analysis results show that ( Figure 10 b) The experimental results of other control groups (III~VI) showed no significant difference, while the viral titer of the experimental group (II) decreased by 100%, and the antiviral efficiency reached 100%.

[0048] The target viruses mentioned above are Zika virus, dengue virus, and chikungunya virus. The inactivation effect of Zika virus is as follows: Figure 10 As shown in the experimental results, the plaque results of the inactivation effects of dengue virus and chikungunya virus are as follows. Figure 11 and Figure 12 .

[0049] Since viral infection of host cells causes apoptosis and a significant decrease in cell viability, but inactivation of the virus does not affect host cell viability, further research was conducted on the viability of host cells under different conditions. It was found that with the extension of reaction time, the cell viability of groups III-VI gradually decreased with increasing culture time, reaching below 30% by day 5, indicating that apoptosis occurred after infection with live virus. In contrast, the cell viability of experimental group II and the Vero cell group without any virus did not change significantly throughout the viral replication cycle, remaining above 90%. These results can be verified through… Figure 10 c. Experimental results explain that calcein yellow-green and pyridine iodide can be used to distinguish between live and dead cells. Calcein yellow-green indicates live cells, which emit green fluorescence, while pyridine iodide indicates dead cells. When cells undergo apoptosis, pyridine iodide can enter the apoptotic cells and embed itself into the cell's DNA, emitting red fluorescence. Therefore, after co-staining cells with these two indicators, live and dead cells are distinguished by the emitted fluorescence color. Figure 10 As shown in d, when the cells treated above were co-stained with calcium yellow-green and pyridine iodide, red fluorescence was clearly observed in groups III to VI, indicating that the cells underwent apoptosis. In contrast, the staining results of group II cells were all green fluorescence, indicating that the cells had a high viability rate. This was mainly because the inactivated virus could not infect the host cells, thus causing the host cells to undergo apoptosis.

[0050] As can be seen from the above embodiments, the integrated detection and inactivation analysis method provided by this invention is simple and rapid to operate, has strong anti-interference capabilities and high sensitivity, and requires no instruments. Simultaneously, it can achieve efficient virus inactivation during detection, effectively reducing the exposure risk to operators, preventing secondary pollution of the environment by the virus, and preventing the risk of secondary virus transmission. This invention provides a new strategy for the rapid detection of low virus concentrations and the suppression of biosafety risk transmission.

[0051] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for the integrated detection and inactivation of arboviruses, characterized in that: Includes the following steps: Step 1: React biotinylated single-stranded DNA with heme to obtain biotin-labeled DNA enzyme; Step 2: Modify the nanospheres containing carboxyl groups with polyclonal antibodies against the target virus and streptavidin molecules to obtain dual-labeled nanospheres; wherein, the polyclonal antibodies against the target virus and streptavidin are modified on the surface of the nanospheres by covalent coupling reaction between the carboxyl groups on the surface of the nanospheres and the amino groups of the protein. Step 3: The sample to be tested is incubated with the monoclonal antibody against the virus to be tested for the first time, followed by separation and washing to obtain the first complex; the double-labeled nanospheres are incubated with the first complex for the second time, followed by separation and washing to obtain the second complex; biotin-labeled DNase is added to the second complex for the third incubation, followed by separation and washing to obtain the third complex; fluorescent red and hydrogen peroxide are added to the third complex for the fourth incubation. The oxidation product of the fluorescent red after being catalyzed and oxidized by the DNase is halogenated. Halogenated has aggregation-induced emission and photodynamic activity. If significant fluorescence is present, it is determined that the sample to be tested contains the virus to be tested. Step four: Irradiate the incubated system under a light source. Use the oxidation product generated in step three as a photosensitizer to generate reactive oxygen species under the light source to inactivate the virus to be tested. The biotin-labeled DNA enzyme has peroxidase catalytic activity.

2. The integrated detection and inactivation method according to claim 1, characterized in that: The virus to be tested is one of Zika virus, dengue virus, and chikungunya virus; If the virus to be tested is Zika virus, the monoclonal antibody against the virus to be tested is an antibody against Zika virus gE protein, and the polyclonal antibody against the virus to be tested is an antibody against Zika gE protein. If the virus to be tested is dengue virus, the monoclonal antibody against the virus to be tested is an anti-dengue virus gE antibody, and the polyclonal antibody against the virus to be tested is an anti-dengue virus gE antibody. If the virus to be tested is Chikungunya virus, the monoclonal antibody against the virus to be tested is Chikungunya virus E1 antibody, and the polyclonal antibody against the virus to be tested is Chikungunya virus E1 protein antibody.

3. The integrated detection and inactivation method according to claim 1, characterized in that: The biotinylated single-stranded DNA is a nucleic acid that is biotin-modified and rich in the base G.

4. The integrated detection and inactivation method according to claim 1, characterized in that: In step one, the biotinylated single-stranded DNA is first heated to 65-90°C, the temperature range for the reaction with heme is 20-30°C, and the pH range for the reaction is 7.0-7.

8.

5. The integrated detection and inactivation method according to claim 1, characterized in that: In step two, nanospheres containing carboxyl-modified groups, polyclonal antibodies against the target virus, and streptavidin are incubated at a temperature of 20-37°C for more than 4 hours to obtain dual-labeled nanospheres simultaneously labeled with polyclonal antibodies against the target virus and streptavidin.

6. The integrated detection and inactivation method according to claim 1, characterized in that: In step two, the mass ratio of polyclonal antibodies against the virus to be tested to streptavidin ranges from 0.5:1 to 6:

1.

7. The integrated detection and inactivation method according to claim 1, characterized in that: In step three, the sample to be tested is co-incubated with the enzyme-linked immunosorbent assay (ELISA) plate containing the monoclonal antibody against the virus to be tested at a temperature of 37°C for more than 2 hours. After washing the ELISA plate, it is blocked and washed again to obtain the first complex.

8. The integrated detection and inactivation method according to claim 1, characterized in that: In step three, the temperature for the second incubation is 25~37℃ and the incubation time is greater than 1 hour; the temperature for the third incubation is 25~37℃ and the incubation time is greater than 0.5 hours; and the temperature for the fourth incubation is 20~30℃ and the incubation time is greater than 0.5 hours.

9. The integrated detection and inactivation method according to claim 1, characterized in that: In step three, quantitative detection of the virus to be tested is performed at a wavelength of 590 nm.

10. The integrated detection and inactivation method according to claim 1, characterized in that: The light source is white light, and the irradiation time is 1 to 60 minutes.

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