Activatable signal amplification detection technology based on fluorescent dye tyramine and application of activatable signal amplification detection technology in nucleocapsid protein detection

Abstract

The invention provides an activatable signal amplification detection technology based on fluorescent dye tyramine and application of the activatable signal amplification detection technology in nucleocapsid protein detection. The problems of insufficient detection sensitivity, poor specificity, loud background noise and the like in a traditional tyramine signal amplification technology are solved by adopting a dual amplification detection technology (combining with an aptamer or an antibody to improve specificity and utilizing tyramine to realize signal amplification) capable of activating tyramine signal amplification and ROS response; and the detection difficulty caused by the own characteristics of the nucleocapsid protein is avoided. According to the technology, the sensitivity, specificity and accuracy of detection are remarkably improved, different virus nucleocapsid proteins can be effectively distinguished, background noise is reduced, a reliable method is provided for SARS-CoV-2 nucleocapsid protein detection, meanwhile, a basis is provided for researching the influence of different structures on the detection effect, and the technology has wide application prospects in the fields of clinical diagnosis and scientific research.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent compounds, and in particular to an activatable signal amplification detection technology based on the fluorescent dye tyramide and its application in nucleocapsid protein detection. Background Art

[0002] Tyramide signal amplification (TSA) is widely used in antigen imaging in tissue samples due to its significant signal enhancement effect, especially low-abundance biomarkers. Traditional tyramide signal amplification technology is usually based on the binding of horseradish peroxidase (HRP)-labeled antibodies to target proteins, and a large amount of free fluorescein tyramide (FT) is covalently coupled to the tyrosine residues of the protein under HRP catalysis, thereby covalently labeling a large amount of fluorescein on the protein to achieve fluorescence signal amplification. For example, in the invention patent application with application publication number CN103513033A and invention name "A visual detection method for Staphylococcus aureus based on tyramide signal amplification technology and aptamer recognition", tyramide signal amplification technology is also used. However, the comparative document is based on the combination of aptamer recognition, biotin-avidin system and tyramide signal amplification to achieve the detection of Staphylococcus aureus. Although it can accurately identify Staphylococcus aureus, under the traditional tyramide signal amplification technology strategy, fluorescein tyramide is in the "Always On" state, resulting in large background noise, which inevitably affects the accuracy and sensitivity of traditional TSA, and makes the reliability of the test results questionable.

[0003] Nucleocapsid protein is an important component of the virus structure, such as the nucleocapsid protein of the SARS-CoV-2 virus. Because it plays a key role in the virus life cycle, its accurate detection is of great significance for the diagnosis and prevention of the disease. There are various methods for detecting nucleocapsid protein in the prior art, including some methods based on immunoassay. However, the nucleocapsid protein itself has some characteristics, such as complex structure, the existence of multiple isomers, and the content in the sample may be low. These characteristics make the existing detection methods face many challenges. Traditional detection methods often have difficulty in accurately identifying nucleocapsid proteins in complex biological samples, and are limited by the sensitivity and specificity of the detection, resulting in unsatisfactory detection results.

[0004] Therefore, it is urgent to find a new activatable signal amplification detection technology based on the fluorescent dye tyramide, and prepare a new fluorescent dye tyramide through a new synthesis method. This fluorescent dye tyramide has the characteristics of simple synthesis method, mild conditions, high yield, and is suitable for large-scale production. In the detection of SARS-CoV-2 nucleocapsid protein, it can significantly reduce background noise, enhance fluorescence signal, and "turn on" fluorescence signal through activated fluorescent dye tyramide, thereby improving the accuracy and sensitivity of detection. The detection specificity can be significantly improved by combining multiple nucleic acid aptamers or antibodies that bind to the antigen to be tested at different sites, thereby achieving efficient and accurate detection of the antigen to be tested. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides an activatable signal amplification detection technology based on fluorescent dye tyramide and its application in nucleocapsid protein detection. First, by incubating beads modified with nucleocapsid protein and antibody, the nucleocapsid protein can be specifically bound to the surface of the beads, providing a stable basis for subsequent detection. Then, the above-mentioned incubated complex is co-incubated with HRP-labeled recognition molecules to form a recognition molecule sandwich structure for detecting nucleocapsid protein. The recognition molecules include nucleic acid aptamers or antibodies, etc., which can specifically recognize different epitopes on nucleocapsid protein. The multi-site specific recognition method greatly improves the specificity of detection. Then, newly synthesized fluorescent dye tyramide and H2O2 are added. Under the catalytic action of the HRP-labeled recognition molecules, H2O2 can activate the fluorescent dye tyramide, so that it produces a signal amplification effect, and realizes the enrichment of tyramide signals. The signal amplification mechanism is based on the dual amplification detection technology of activatable tyramide signal amplification and ROS response, which greatly improves the sensitivity of detecting the target by cleverly utilizing the characteristics of the fluorescent dye tyramide and the specific binding of the recognition molecule to the nucleocapsid protein. At the same time, the present invention can accurately distinguish SARS-CoV-2 nucleocapsid protein by constructing different formulas for detection comparison, and selecting a variety of viral nucleocapsid proteins for specificity verification tests, which significantly improves the specificity of SARS-CoV-2 nucleocapsid protein detection. Therefore, the present invention achieves signal amplification, improves the accuracy, sensitivity and specificity of detection in nucleocapsid protein detection through a unique mechanism of action, and provides an efficient and accurate method for the detection of SARS-CoV-2 nucleocapsid protein.

[0006] In order to achieve the above object, the present invention adopts the following scheme:

[0007] On the one hand, the present invention provides an activatable signal amplification system based on the fluorescent dye tyramide, characterized in that the activatable signal amplification system includes a fluorescent dye tyramide, and the fluorescence of the fluorescent dye tyramide can be activated only in the presence of ROS, thereby realizing activated tyramide signal amplification.

[0008] Furthermore, the fluorescent dye tyramine includes the structural formulas shown in formula (I) and formula (II):

[0009]

[0010] Among them, R1 or R2 includes any one or more of hydrogen atoms, fluorine atoms, chlorine atoms, bromine atoms or iodine atoms; R3, R5 or R6 includes any one or more of hydrogen atoms or chlorine atoms; R4 includes any one or more of hydrogen atoms, chlorine atoms or nitro groups.

[0011] Since the traditional tyramide signal amplification technology has problems such as large background noise, poor detection sensitivity and specificity, and the characteristics of the nucleocapsid protein itself are complex in structure, multiple isomers, and the content in the sample may be low, which makes detection difficult, the present invention proposes a new recognition molecule sandwich structure, and optimizes the original tyramide signal amplification system in many aspects to form a new detection mechanism based on fluorescent dye tyramide that can activate signal amplification. The mechanism is as follows: (1) Recognition molecule sandwich structure: by incubating the nucleocapsid protein with antibody-modified beads, the nucleocapsid protein can be specifically bound to the surface of the beads, and then co-incubated with HRP-labeled recognition molecules (nucleic acid aptamers or antibodies) to form a recognition molecule sandwich structure. This structure uses the characteristics of nucleic acid aptamers and antibodies that can specifically recognize different epitopes on nucleocapsid proteins, greatly improving the specificity of detection; (2) Signal amplification mechanism: using activatable tyramide signal amplification and ROS response dual amplification detection technology. In this mechanism, newly synthesized fluorescent dye tyramide and H2O2 are added. Under the catalysis of HRP-labeled recognition molecules, H2O2 activates the fluorescent dye tyramide, causing it to amplify the signal and achieve the enrichment of tyramide signals, thereby improving the accuracy and sensitivity of target detection.

[0012] Therefore, in some embodiments, a new fluorescent dye tyramine is first synthesized by a new synthesis method, and its structural type is shown in formula (I) and formula (II). In formula (I), R1 or R2 is a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom; R3, R5 or R6 is a hydrogen atom or a chlorine atom; R4 is a hydrogen atom, a chlorine atom or a nitro group, and its structural formula includes any one of formula (I-1) to formula (I-6). In formula (II), R1 or R2 is a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom; R3, R5 or R6 is a hydrogen atom or a chlorine atom; R4 is a hydrogen atom, a chlorine atom or a nitro group, and its structural formula includes formula (II-1).

[0013] The structural feature makes the newly synthesized fluorescent dye tyramide of the present invention have an activatable characteristic. When there are recognition molecules labeled with HRP and H2O2, the fluorescent dye tyramide can be activated, thereby covalently coupling with the tyrosine residues on the nucleocapsid protein to play a role in signal amplification. This activatable characteristic avoids the defect of large background noise caused by the "Always On" state of fluorescein tyramide in traditional tyramide signal amplification technology. Through the activation mechanism, the fluorescent dye tyramide only generates a signal under specific conditions, greatly reducing the background noise, improving the signal-to-noise ratio of the detection signal, and then improving the accuracy and sensitivity of the detection, for nucleocapsid protein detection provides a more reliable method.

[0014] Furthermore, the structural formula represented by formula (I) includes any one of formula (I-1) to formula (I-6):

[0015]

[0016] Furthermore, the structural formula represented by formula (II) includes formula (II-1):

[0017]

[0018] In some embodiments, in order to clarify the detection effect of fluorescent dye tyramine with different structures on SARS-CoV-2 nucleocapsid protein, the amplification effect, sensitivity, specificity and accuracy of seven different structures of fluorescent dye tyramine of formula (I-1) to formula (I-6) and formula (II-1) in the detection of SARS-CoV-2 nucleocapsid protein were compared and tested. The experimental results show that all structures of fluorescent dye tyramine in formula C (the formula of fluorescent dye tyramine with the best structure of the present invention) show more obvious fluorescence shift than formula A (FITC-labeled aptamer formula only). Among them, the fluorescence shift of formula (I-1), (I-2), formula (I-3), formula (I-4) and formula (II-1) in formula C is the most obvious, indicating that these structures may have better performance in signal amplification and detection sensitivity. Although the fluorescence shift of formula (I-5) and formula (I-6) is slightly smaller, it still has a significant shift compared with formula A, and the fluorescence background is moderate, and it also has a good signal amplification effect and detection sensitivity. This may be because the fluorescent dye tyramide of the structural type of formula (I) has a specific combination of substituents and a spatial structure, which enables it to play a more effective role in binding to the nucleocapsid protein and in the signal amplification process. The fluorescent dye tyramide of the structural type of formula (II) also has a similar chemical structure and active site, which enables it to participate in the signal amplification process better, so both show good signal amplification and detection sensitivity characteristics.

[0019] The fluorescent dye tyramide in formula C can be clearly distinguished from formula A (almost no deviation) when there is nucleocapsid protein, and when there is no nucleocapsid protein, the background fluorescence of most structures of fluorescent dye tyramide (formula (I-1), formula (I-2), formula (I-3), formula (I-4) and formula (II-1) etc.) in formula C is very low. Combined with the signal-to-noise ratio analysis, the fluorescent dye tyramide of these structures performs better in specificity and accuracy. Although the fluorescence background of formula (I-5) and formula (I-6) is slightly higher than that of other structures, it is still significantly lower than that of formula A, so it also has good specificity and accuracy.

[0020] Therefore, it can be seen that the new fluorescent dye tyramide prepared by the present invention has good detection performance, and different structures show different degrees of advantages in detecting SARS-CoV-2 nucleocapsid protein, which can provide a basis for selecting fluorescent dye tyramide with suitable structure according to specific needs in practical applications.

[0021] On the other hand, the present invention provides a method for preparing the fluorescent dye tyramide formula (I) in the activatable signal amplification system as described in any one of the above technical solutions, comprising the following steps:

[0022] (1) Using a fluorescein derivative as a raw material, sodium borohydride as a reducing agent, and tetrahydrofuran as a solvent, the reaction was carried out at room temperature to obtain compound III;

[0023] (2) Using N-methyltyramine and TBSCl as raw materials, imidazole as a base, and tetrahydrofuran as a solvent, the reaction was carried out at room temperature to obtain compound IV;

[0024] (3) Compound III and imidazole were dissolved in tetrahydrofuran, TBSCl was added dropwise at 0°C, the temperature was slowly raised to room temperature, and the mixture was stirred to obtain compound V;

[0025] (4) Compounds III and V were dissolved in DMF, triethylamine and HOBT were added, and then triethylamine was added, and the mixture was stirred at room temperature overnight to obtain compound VI;

[0026] (5) Compound VI was dissolved in THF, tetrabutylammonium fluoride was added under nitrogen atmosphere, and the mixture was stirred at room temperature for 2 hours to obtain compound VII, which is the fluorescent dye tyramide formula (I).

[0027] Furthermore, in step (1), the molar ratio of the fluorescein derivative to sodium borohydride is 1:6; in step (2), the molar ratio of N-methyltyramine to TBSCl is 1:1.2; in step (3), the molar ratio of compound III to TBSCl is 1:3; in step (4), the molar ratio of compound V to III is 1:1.2; in step (5), the molar ratio of compound VI to TBAF is 1:5, and step (5) is carried out under nitrogen protection throughout.

[0028] In another aspect, the present invention provides a method for preparing the fluorescent dye tyramide formula (II) in the activatable signal amplification system as described in any one of the above technical solutions, comprising the following steps:

[0029] (1) Using rhodamine as a raw material, sodium borohydride as a reducing agent, and tetrahydrofuran as a solvent, react at room temperature to obtain compound VIII;

[0030] (2) Compounds VIII and IV were dissolved in DMF, triethylamine and HOBT were added, and then triethylamine was added, and the mixture was stirred at room temperature overnight to obtain compound VIIII;

[0031] (3) Compound VIIII was dissolved in THF, tetrabutylammonium fluoride was added under nitrogen atmosphere, and the mixture was stirred at room temperature for 2 hours to obtain compound X, which is the fluorescent dye tyramide formula (II).

[0032] In another aspect, the present invention provides a biomolecule based on an activatable signal amplification strategy, wherein the biomolecule includes the fluorescent dye tyramide, and the fluorescent dye tyramide includes the fluorescent dye tyramide in any activatable signal amplification system in the above technical solutions.

[0033] Furthermore, the biomolecules also include any one or more combinations of antibodies, nucleic acid aptamers, polypeptides, and small molecules that specifically recognize antigens.

[0034] Furthermore, horseradish peroxidase is used to label nucleic acid aptamers, the nucleic acid aptamers are nucleic acid sequences that can specifically recognize the antigen to be detected, and the nucleic acid sequences of the nucleic acid aptamers are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.

[0035] In some embodiments, in order to further improve the accuracy, sensitivity and specificity of nucleocapsid protein detection, the present invention is also optimized from the perspective of nucleic acid aptamers.

[0036] In terms of nucleic acid aptamers, the present invention uses nucleic acid aptamers that can specifically identify nucleocapsid proteins, and its nucleic acid sequence is designed and screened, and has the ability to specifically bind to nucleocapsid proteins. First, nucleic acid aptamers can bind to specific sites on nucleocapsid proteins, and then react with fluorescent dye tyramide and H2O2 through HRP-labeled nucleic acid aptamers. In this process, the specific binding effect of nucleic acid aptamers makes the signal amplification process more accurately targeted at nucleocapsid proteins. At the same time, by constructing different formulas for detection comparison (such as formula 1, formula 2 and formula 3), it can be seen that the synergistic effect of nucleic acid aptamers and fluorescent dye tyramide and HRP can improve the sensitivity and specificity of detection to varying degrees. For example, in formula 3, after the HRP-labeled nucleic acid aptamer targets the nucleocapsid protein, the "Turn on" molecule T-DCFH is used to achieve fluorescent signal amplification, which shows higher sensitivity and specificity when detecting nucleocapsid proteins.

[0037] The traditional tyramide signal amplification technology mainly relies on the combination of antibodies and nucleocapsid proteins and the subsequent fluorescein tyramide amplification process, and lacks a highly specific recognition molecule such as a nucleic acid aptamer. The present invention avoids the problem of poor detection sensitivity and specificity caused by the lack of specific recognition molecules in the traditional technology by using nucleic acid aptamers, and at the same time utilizes the synergistic effect of nucleic acid aptamers with fluorescent dyes tyramide and HRP to further improve the sensitivity and specificity of detection and reduce background noise.

[0038] Furthermore, the antibody that specifically recognizes the antigen is an antibody that specifically recognizes the nucleocapsid protein.

[0039] Furthermore, the concentration of the horseradish peroxidase is 2.0 μg / mL.

[0040] In some embodiments, in order to screen out the amplification efficiency of T-DCFH at different HRP concentrations when hydrogen peroxide is applied, and to find the HRP concentration condition that makes the amplification efficiency the strongest, the present invention conducts corresponding exploratory experiments, and the experimental results show that when the HRP concentration is 2.0 μg / mL, the fluorescence value is the largest, the amplification efficiency is the strongest, and there is basically no fluorescence when HRP is not present. In addition, the fluorescence signals all increase rapidly, eventually reaching saturation values, and showing good concentration dependence. Therefore, it can be seen that only in the presence of HRP can T-DCFH be effectively activated and produce fluorescence signals, and the fluorescence signal intensity shows a good dependence on the concentration of HRP.

[0041] Furthermore, the concentration of the nucleic acid aptamer coupled to horseradish peroxidase is 20 nM.

[0042] In some embodiments, in order to screen out the amplification efficiency of T-DCFH at different HRP concentrations of nucleic acid aptamer conjugates under the action of hydrogen peroxide, and find the HRP concentration conditions of nucleic acid aptamer conjugates with the strongest amplification efficiency, the present invention has carried out corresponding exploration experiments, and the experimental results show that when the concentration of nucleic acid aptamer conjugate HRP is 20nM, the fluorescence value is the largest, the amplification efficiency is the strongest, and there is basically no fluorescence when nucleic acid aptamer HRP does not exist. In addition, the fluorescence signal increases rapidly, eventually reaches a saturation value, and shows good concentration dependence. Therefore, it can be seen that in this experiment for the study of different nucleic acid aptamer-HRP conjugates for detecting nucleocapsid protein, the HRP concentration of nucleic acid aptamer conjugates has an important influence on the amplification efficiency of T-DCFH, 20nM is the concentration that maximizes the fluorescence value and has the strongest amplification efficiency, and the fluorescence signal increases rapidly to saturation and has good concentration dependence.

[0043] Furthermore, the concentration of the antibody-coupled horseradish peroxidase is 2.0 μg / mL.

[0044] In some embodiments, in order to screen out the amplification efficiency of T-DCFH at different HRP concentrations of antibody conjugates under the action of hydrogen peroxide, and find the HRP concentration conditions of the antibody conjugate that makes the amplification efficiency the strongest, the present invention conducts corresponding exploratory experiments, and the experimental results show that when the antibody-conjugated HRP concentration is 2.0 μg / mL, the fluorescence value is the largest, the amplification efficiency is the strongest, and there is basically no fluorescence when the antibody-conjugated HRP does not exist. In addition, the fluorescence signals all increase rapidly, eventually reaching saturation values, and showing good concentration dependence. Therefore, it can be seen that in this experiment, in the study of antibodies specifically recognizing SARS-CoV-2 nucleocapsid protein, the antibody-conjugated HRP concentration has a key influence on the amplification efficiency of T-DCFH, 2.0 μg / mL is the concentration that maximizes the fluorescence value and has the strongest amplification efficiency, and the fluorescence signal increases rapidly to saturation and has good concentration dependence, which provides a basis for the reasonable selection of the HRP concentration of the antibody conjugate based on the HRP-T-DCFH detection technology of SARS-CoV-2 nucleocapsid protein.

[0045] In another aspect, the present invention provides an activatable signal amplification detection method based on the fluorescent dye tyramide, wherein the method activates the fluorescent dye tyramide through the biological molecules described in any one of the above technical solutions, thereby specifically identifying the antigen to be detected and achieving signal amplification.

[0046] Furthermore, the detection method comprises the following steps:

[0047] (1) incubating the antigen to be detected and the antibody-modified beads, and then co-incubating with the HRP-labeled aptamer to form a recognition molecule sandwich structure for detecting the antigen to be detected;

[0048] (2) Fluorescent dyes tyramide and H2O2 are then added to achieve enrichment, thereby specifically identifying the antigen to be tested and achieving signal amplification.

[0049] Similarly, in some embodiments of the present invention, in order to improve the sensitivity, specificity and accuracy of the detection of the antigen (nucleocapsid protein) to be tested and overcome the defects of the traditional tyramide signal amplification technology, a new activatable signal amplification detection method based on the fluorescent dye tyramide is proposed. By constructing a recognition molecule sandwich structure combined with the dual amplification technology of activatable tyramide signal amplification and ROS response, it plays a role in specific recognition and signal amplification, has the effect of improving the detection performance and effectively distinguishing different viral nucleocapsid proteins.

[0050] In another aspect, the present invention provides a use of a fluorescent dye tyramide for preparing a preparation for improving the accuracy, sensitivity and specificity of antigen detection, wherein the fluorescent dye tyramide includes the fluorescent dye tyramide in any one of the above technical solutions that can activate the signal amplification system.

[0051] In another aspect, the present invention provides a use of a biomolecule for preparing a preparation for improving the accuracy, sensitivity and specificity of antigen detection, wherein the biomolecule is as described in any one of the above technical solutions.

[0052] In some embodiments, in order to verify that the activatable signal amplification detection technology based on fluorescent dye tyramide of the present invention (including the use of a newly synthesized fluorescent dye tyramide and a biological molecule with a recognition molecule sandwich structure) can significantly improve the detection accuracy, sensitivity and specificity of the antigen (nucleocapsid protein) to be tested, verification experiments were carried out.

[0053] The results of the accuracy and sensitivity validation test showed that: by constructing three different formulations (Formula 1: triple FITC-labeled aptamer; Formula 2: triple aptamer-HRP with "Always on" FT; Formula 3: triple aptamer-HRP with "Turn on" T-DCFH), flow cytometry analysis of different concentrations of SARS-CoV-2 nucleocapsid protein was performed. Almost no fluorescence shift was observed in Formula 1; there was an obvious fluorescence shift in Formula 2, but the background fluorescence was high in the absence of nucleocapsid protein; in Formula 3, not only was an obvious fluorescence shift observed, but the fluorescence background was also low, which was consistent with the results of quantitative fluorescence analysis. Therefore, it can be seen that Formula 3 (Triple aptamer-HRP with "Turn on" T-DCFH) has high accuracy and sensitivity in detecting nucleocapsid protein, can accurately detect in low-concentration nucleocapsid protein samples, and has a low fluorescence background, which improves the accuracy and reliability of the detection.

[0054] The results of the specificity verification test show that the deviation of influenza A H3N2, HCOv-Hku1, Hcov-229E and MERS-Cov is very small, while the deviation of SARS-CoV-2 nucleocapsid protein is obvious, and the value of SARS-CoV-2 nucleocapsid protein is higher than that of the other four virus-related nucleocapsid proteins. At the same time, the quantitative analysis of the signal-to-noise ratio shows that the signal-to-noise ratio of the SARS-CoV-2 group is about 7, which is significantly higher than that of the other four groups. Therefore, it can be seen that the detection technology of the present invention has a high specificity for SARS-CoV-2 nucleocapsid protein, and can effectively distinguish SARS-CoV-2 nucleocapsid protein from the nucleocapsid proteins of the other four infectious viruses.

[0055] The beneficial effects of the present invention are:

[0056] 1. The present invention provides an activatable signal amplification detection technology based on fluorescent dye tyramide and its application in nucleocapsid protein detection. First, a new fluorescent dye tyramide synthesis method is provided, which has the characteristics of mild reaction conditions and high yield, avoids the side reactions and operational complexity that may be caused by harsh conditions, and is suitable for large-scale production. Two main types of fluorescent dye tyramide structural formulas are provided, covering a variety of specific structural forms, which provide a basis for subsequent research on the influence of different structures on the detection effect, and further improve the detection performance.

[0057] 2. The present invention provides a new dual amplification detection technology through activatable tyramide signal amplification and ROS response, which achieves a significant improvement in detection specificity by cleverly combining multiple nucleic acid aptamers or antibodies that bind to nucleocapsid proteins at different sites. Nucleic acid aptamers and antibodies can specifically recognize different epitopes on nucleocapsid proteins, and this multi-site binding method increases the accuracy and specificity of detection. At the same time, the use of fluorescent dye tyramide to achieve signal amplification plays a key role in improving detection sensitivity.

[0058] At the same time, the detection technology of the present invention effectively overcomes many defects of traditional tyramide signal amplification technology. In traditional technology, fluorescein tyramide is in the "Always On" state, resulting in large background noise, which seriously affects the accuracy and sensitivity of the detection. The detection technology of the present invention uses an activatable signal amplification mechanism, and the fluorescent dye tyramide is activated to generate a signal only under specific conditions, thereby greatly reducing the background noise and improving the signal-to-noise ratio of the detection signal, providing a more reliable, accurate and sensitive method for the detection of SARS-CoV-2 nucleocapsid protein.

[0059] By combining multiple nucleic acid aptamers or antibodies that bind to the nucleocapsid protein at different sites to improve detection specificity, and using the fluorescent dye tyramide to achieve signal amplification, the accuracy, sensitivity and specificity of detection are effectively improved, overcoming the defects of traditional tyramide signal amplification technology such as large background noise, and providing a more reliable method for SARS-CoV-2 nucleocapsid protein detection.

[0060] 3. In the existing nucleocapsid protein detection methods, the detection faces many difficulties due to the complex structure of the nucleocapsid protein itself, the existence of multiple isomers, and the low content in the sample. Traditional detection methods often rely on a single recognition mechanism, which makes it difficult to accurately identify nucleocapsid proteins in complex biological samples. At the same time, these methods have limitations in signal amplification and large background noise, which leads to poor sensitivity and specificity of detection and cannot meet the needs of practical applications.

[0061] Therefore, the present invention first proposes a new method for synthesizing fluorescent dye tyramide, and synthesizes a new fluorescent dye tyramide with specific structure and performance. Then, a new principle of activatable signal amplification detection technology based on fluorescent dye tyramide is adopted. Through the above-mentioned dual amplification mechanism and the specific binding mode with nucleocapsid protein, the problems existing in the existing detection method are well solved. Finally, the sensitivity and specificity of the detection are significantly improved, the background noise is reduced, and the nucleocapsid protein can be accurately detected in complex biological samples, which provides an efficient and reliable solution for the detection of nucleocapsid protein, and has important application value and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The figure is a flow chart of the fluorescent dye tyramide-based activatable signal amplification detection technology for nucleocapsid protein detection.

[0063] Figure 2 The schematic diagram of the structure of the fluorescent dye in the fluorescent dye tyramine provided by the present invention.

[0064] Figure 3 The present invention provides two structural formulas of the fluorescent dye tyramine (Formula (I) and Formula (II); wherein: R1 or R2 is a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom; R3, R5 or R6 is a hydrogen atom or a chlorine atom; R4 is a hydrogen atom, a chlorine atom or a nitro group).

[0065] Figure 4 The specific structural formulas of the fluorescent dye tyramine shown in formula (I) (Formula (I-1) to Formula (I-6)).

[0066] Figure 5 It is the specific structural formula (Formula (II-1)) of the fluorescent dye tyramine shown in Formula (II).

[0067] Figure 6 The schematic diagram of the synthesis of compound III in the preparation process of fluorescent dye tyramide formula (I).

[0068] Figure 7 Schematic diagram of the synthesis of compound IV during the preparation of fluorescent dye tyramide formula (I).

[0069] Figure 8 Schematic diagram of the synthesis of compound V during the preparation of fluorescent dye tyramide formula (I).

[0070] Fig. 9 The schematic diagram of the synthesis of compound VI during the preparation of fluorescent dye tyramide formula (I).

[0071] Fig.10 Schematic diagram of the synthesis of fluorescent dye tyramine formula (I) (from compound VII).

[0072] Fig.11 The schematic diagram of the synthesis of compound VIII in the preparation process of fluorescent dye tyramide formula (II).

[0073] Fig.12 The schematic diagram of the synthesis of compound VIIII during the preparation of fluorescent dye tyramide formula (II).

[0074] Fig.13 Schematic diagram of the synthesis of fluorescent dye tyramide formula (II) (compound X).

[0075] Fig.14 The present invention provides an activatable "Turn On" type fluorescent molecule based on the fluorescent dye tyramine: the specific structural formula of T-DCFH.

[0076] Fig.15 This is the synthetic route of T-DCFH.

[0077] Fig.16 This is a diagram showing the verification of the fluorescence properties of T-DCFH and the measurement results of the excitation wavelength and emission wavelength.

[0078] Fig.17 a is a schematic diagram of T-DCFH-based activatable fluorescence amplification for HE4 protein detection in ELISA plate.

[0079] Fig.17 b shows the fluorescence “Turn On” based on T-DCFH by adding different concentrations of HE4 protein (including 62.5, 125, 250, 500, 1000 and 2000 pg / mL) to calculate the fluorescence intensity.

[0080] Fig.17 c is a linear relationship in the range of 0 to 2,000 pg / mL.

[0081] Fig.18 a is the verification that HRP can activate fluorescence amplification of T-DCFH.

[0082] Fig.18 b is a graph showing the change in dynamic fluorescence signal by adding different concentrations of HRP (2.0 μg / mL, 1.0 μg / mL, 0.5 μg / mL, and 0.25 μg / mL).

[0083] Fig.19 This is a graph showing the effect of HRP concentrations of different antibody conjugates on T-DCFH amplification efficiency.

[0084] Fig. 20 This is a graph showing the effect of HRP concentrations of different aptamer conjugates on T-DCFH amplification efficiency.

[0085] Fig.21 a is formulation 1: triple FITC-labeled aptamer.

[0086] Fig.21 b, Formulation 2: Triple Aptamer-HRP with “Always on” FT.

[0087] Fig.21 c, Formulation 3: Triple Aptamer-HRP with "Turn on" T-DCFH.

[0088] Fig.21 d is flow cytometry analysis of different concentrations of SARSCoV-2 nucleocapsid protein in formulation 1 ranging from 0, 5, 10, 50, 100, 200, 400, 800 to 1000 ng / mL.

[0089] Fig.21 e is flow cytometric analysis of different concentrations of SARSCoV-2 nucleocapsid protein in formulation 2 ranging from 0, 5, 10, 50, 100, 200, 400, 800 to 1000 ng / mL.

[0090] Fig.21 f is flow cytometry analysis of different concentrations of SARSCoV-2 nucleocapsid protein in formulation 3 ranging from 0, 5, 10, 50, 100, 200, 400, 800 to 1000 ng / mL.

[0091] Fig.21 g is the statistical fluorescence intensity of the three formulations.

[0092] Fig.21 h is the fluorescence quantitative analysis of the three formulations, showing that the signal-to-noise ratio of the aptamer-HRP / T-DCFH group is the highest, and F0 represents the fluorescence intensity without the target protein. All data are mean ± SD, n = 3.

[0093] Fig. 22 a is the modification process of nucleic acid aptamer with HRP through chemical covalent coupling.

[0094] Fig. 22 b is Native-PAGE analysis of aptamer-HRP conjugates (including N35-HRP, N10-HRP and N2-62-HRP). Lane 1, N35; Lane 2, unpurified N35-HRP; Lane 3, purified N35-HRP; Lane 4, N10; Lane 5, unpurified N10-HRP; Lane 6, purified N10-HRP; Lane 7, N2-62; Lane 8, unpurified N2-62-HRP; Lane 9, purified N2-62-HRP; Lane L, DNA ladder.

[0095] Fig.23 a Flow cytometric analysis of 1000 ng / mL nucleocapsid protein of infectious viruses including SARS-CoV-2, influenza A H3N2, HCOv-Hku1, Hcov-229E and MERS-Cov.

[0096] Fig.23 b corresponds to Fig.23 Fluorescence intensity statistics of a.

[0097] Fig.23 c is the quantitative analysis of signal-to-noise ratio, showing that the signal-to-noise ratio of the SARS-CoV-2 group is higher than that of influenza A H3N2, HCOv-Hku1, Hcov-229E and MERS-CoV groups; F0 represents the fluorescence intensity without target protein, and FBeads represents the fluorescence intensity of beads only. All data are mean ± sd, n = 3.

[0098] Fig.23 d is confocal laser microscopy imaging showing that the fluorescence signal of preparation 3 incubated with 1000 ng / mL SARS-CoV-2 nucleocapsid protein is the highest. The scale bar is 5 μm.

[0099] Fig.23 e: Quantitative analysis of the slices (white arrows) imaged in confocal laser microscopy. DETAILED DESCRIPTION

[0100] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be pointed out that the embodiments described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on the present invention.

[0101] Example 1 Synthesis of fluorescent dye tyramine

[0102] In the prior art, the detection of nucleocapsid protein faces many challenges. Traditional detection methods have problems such as insufficient sensitivity, difficulty in accurate detection in low-concentration samples, and high detection limits; poor specificity, and easy interference by other similar proteins or substances in the sample, resulting in false positive or false negative results; signal amplification technology has limitations, such as the traditional tyramide signal amplification technology in which fluorescein tyramide is in the "Always On" state, large background noise, affecting accuracy and sensitivity, and the reliability of the detection results is questioned. Based on the above shortcomings, the present invention proposes a new activatable signal amplification detection technology based on fluorescent dye tyramide for the detection of nucleocapsid protein. The technology is based on the dual amplification detection technology of activatable tyramide signal amplification and ROS response, by synthesizing a new fluorescent dye tyramide that can only be activated by fluorescence in the presence of ROS, combining nucleic acid aptamers or antibodies to improve specificity, and using tyramide to achieve signal amplification, thereby solving the problems of insufficient detection sensitivity, poor specificity, and large background noise in traditional tyramide signal amplification technology, as well as the detection difficulties caused by the characteristics of nucleocapsid protein itself.

[0103] The detection technology firstly involves a new synthesis method of fluorescent dye tyramide, which has the characteristics of mild reaction conditions and high yield, and is suitable for large-scale production. Then, the dual amplification detection technology of activatable tyramide signal amplification and ROS response is used to improve the detection specificity by combining multiple nucleic acid aptamers or antibodies that bind to the nucleocapsid protein at different sites, and the fluorescent dye tyramide is combined to achieve signal amplification, thereby improving the accuracy and sensitivity of the detection.

[0104] The present invention incubates nucleocapsid protein and antibody-modified beads so that the nucleocapsid protein can be specifically bound to the surface of the beads, and then co-incubated with HRP-labeled recognition molecules to form a recognition molecule sandwich structure for detecting nucleocapsid protein, and then fluorescent dye tyramide T-DCFH and H2O2 are added to achieve enrichment. Under the catalytic action of the HRP-labeled recognition molecules, H2O2, as a kind of ROS, can activate the fluorescent dye tyramide. The activated fluorescent dye tyramide can produce a signal amplification effect, achieve the enrichment of tyramide signals, and achieve enhanced specific detection. Its specific principle is based on the special structure and properties of the newly synthesized fluorescent dye tyramide of the present invention. It is only activated under specific conditions (there are ROS and there are HRP-labeled recognition molecules catalyzed), avoiding the defect of large background noise caused by the fluorescein tyramide in the "Always On" state in the traditional tyramide signal amplification technology. The activated fluorescent dye tyramide can be covalently coupled to the tyrosine residues on the nucleocapsid protein, thereby playing a role in signal amplification and improving the sensitivity of detecting the target. And it was verified in a nucleocapsid protein detection system. The specific process is as follows Figure 1 shown.

[0105] The fluorescent dye in the fluorescent dye tyramine provided by the present invention can be any one of 2',7'-dichlorodihydrofluorescein, 2',7'-difluorodihydrofluorescein, 2',4',5',7'-tetrabromodihydrofluorescein, 2',4',5',7'-tetraiododihydrofluorescein, tetrachlorodihydrofluorescein, nitrodihydrofluorescein, and rhodamine B, such as Figure 2 . (All fluorescent molecules activated in response to ROS).

[0106] The present invention provides two structural formulas of fluorescent dye tyramine and corresponding synthesis methods thereof, wherein the structural formulas are respectively shown in formula (I) and formula (II) ( Figure 3 ):

[0107]

[0108] Wherein: R1 or R2 is a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom; R3, R5 or R6 is a hydrogen atom or a chlorine atom; R4 is a hydrogen atom, a chlorine atom or a nitro group.

[0109] The fluorescent dye tyramine represented by the formula (I) has a structural formula including any one of formula (I-1) to formula (I-6) ( Figure 4 ):

[0110]

[0111] The fluorescent dye tyramine represented by formula (II) has a structural formula including formula (II-1) ( Figure 5 ):

[0112]

[0113] At the same time, the present invention also provides two methods for synthesizing fluorescent dye tyramine, wherein the preparation method of fluorescent dye tyramine formula (I) comprises the following steps:

[0114] (1) Using a fluorescein derivative as a raw material, sodium borohydride as a reducing agent, and tetrahydrofuran as a solvent, a reaction was performed at room temperature to obtain compound III ( Figure 6 ).

[0115]

[0116] (2) Using N-methyltyramine and TBSCl as raw materials, imidazole as a base, and tetrahydrofuran as a solvent, the reaction was carried out at room temperature to obtain compound IV ( Figure 7 ).

[0117]

[0118] (3) The compound III prepared above and imidazole were dissolved in tetrahydrofuran, TBSCl was added dropwise at 0°C, and the mixture was slowly heated to room temperature and stirred for 2 hours to obtain compound V ( Figure 8 ).

[0119]

[0120] (4) Compounds III and V prepared above were dissolved in DMF, triethylamine and HOBT were added, and then triethylamine was added, and the mixture was stirred at room temperature overnight to obtain compound VI ( Fig. 9 ).

[0121]

[0122] (5) The compound VI prepared above was dissolved in THF, tetrabutylammonium fluoride was added under nitrogen atmosphere, and the mixture was stirred at room temperature for 2 hours to obtain compound VII, which is the fluorescent dye tyramine formula (I) ( Fig.10 ).

[0123]

[0124] Wherein, in the step (1), the molar ratio of the fluorescein derivative to sodium borohydride is 1:6; in the step (2), the molar ratio of the N-methyltyramine to TBSCl is 1:1.2; in the step (3), the molar ratio of the compound III to TBSCl is 1:3; in the step (4), the molar ratio of the compound V to III is 1:1.2; in the step (5), the molar ratio of the compound VI to TBAF is 1:5, and the entire step (5) is carried out under nitrogen protection.

[0125] The preparation method of the fluorescent dye tyramide formula (II) comprises the following steps:

[0126] (1) Rhodamine is used as a raw material, sodium borohydride is used as a reducing agent, and tetrahydrofuran is used as a solvent. Compound VIII ( Fig.11 ).

[0127]

[0128] (2) Compounds VIII and IV prepared above were dissolved in DMF, triethylamine and HOBT were added, and then triethylamine was added, and the mixture was stirred at room temperature overnight to obtain compound VIIII ( Fig.12 ).

[0129]

[0130] (3) The compound VIIII prepared above was dissolved in THF, tetrabutylammonium fluoride was added under nitrogen atmosphere, and stirred at room temperature for 2 hours to obtain compound X, which is the fluorescent dye tyramine formula (II) ( Fig.13 ).

[0131]

[0132] In this embodiment, taking the fluorescent dye tyramide formula (I) as an example, R1, R3, R4, R5, and R6 are all hydrogen atoms, and R2 is a chlorine atom. An activatable "Turn On" type fluorescent molecule based on the fluorescent dye tyramide is provided, named: T-DCFH, and its structural formula is shown below ( Fig.14 ):

[0133]

[0134] The specific synthetic route is as follows: Fig.15 ):

[0135]

[0136] The specific synthesis method is as follows:

[0137] (1) First, the fluorescent dye tyramine molecule (Formula 1) was synthesized: 2',7'-dichlorofluorescein (401.2 mg, 1.0 mmol) was placed in a 25 mL round-bottom flask, 4.0 mL of tetrahydrofuran was added to dissolve, and then NaBH4 (6 mmol, 226.98 mg) was added. The mixture was stirred at room temperature for 96 hours, the solvent was removed by vacuum distillation, and column chromatography was purified (MeOH:DCM=1:50) to obtain yellow solid 2',7'-dichlorodihydrofluorescein. The yield was 291.4 mg, and the yield was 73%.

[0138] 1H NMR spectrum(400MHz,DMSO-d6)of DCFH.1H NMR(400MHz,DMSO-d6)δ13.45(br s,1H),10.54(s,2H),7.84(d,J=7.8,1.3Hz,1H),7.52-7.28(m,2H),7.03-6.95(m,3H),6.79(s,2H),6.17(s,1H).ESI:401.00[MH]+,calculated for C20H12Cl2O5:402.01.

[0139] (2) 2',7'-dichlorodihydrofluorescein (1.0mmol, 402.2mg) and imidazole (8.0mmol, 544.0mg) were placed in a 25mL round-bottom flask, 4.0mL of tetrahydrofuran was added to dissolve, and then TBSCl (5mmol, 753.5mg) was slowly added dropwise at 0°C. After the addition was complete, the ice bath was removed and stirred at room temperature for 2h. After the reaction was completed, the reaction solution was washed three times with sodium bicarbonate and brine, the organic phases were combined, dried over sodium sulfate, the solvent was removed under reduced pressure, and the product was purified by column chromatography (EA:PE=1:4). The yield was 253.2mg, and the yield was 40%.

[0140] 1H NMR(400MHz, CDCl3) δ8.06(d,J=8.2Hz,1H),7.42(t,J=7.5,1H),7.34-7.27(m,1H),7.10(d,J=6.8Hz,1 H),6.99(s,2H),6.64(s,2H),6.44(s,1H),1.02(s,18H),0.25(s,12H).ESI:629.20[MH]+,calculated for C32H40Cl2O5Si2:630.20.

[0141] (3) N-methyltyramine (1.9 g, 10.1 mmol) and imidazole (1.4 g, 20.3 mmol) were dissolved in DCM, TBSCl was slowly added dropwise at 0°C, the ice bath was removed, and the mixture was stirred at room temperature for 2 h. The mixture was washed three times with sodium bicarbonate and brine, and the organic phases were combined and dried over sodium sulfate. The product was purified by column chromatography (EA:PE=1:1). The yield was 1.3 g, and the yield was 53%.

[0142] 1H NMR (400MHz, CDCl3) δ6.87(d,J=8.4Hz,2H),6.58(d,J=8.4Hz,2H),2.77-2.61(m,2H),2. 60-2.51(m,2H),2.26(s,3H),0.79(s,9H),-0.00(s,6H).ESI:266.20[M+H]+,calculated for C15H27NOSi:265.19.

[0143] (4) Compounds DCFH-TBS (1 mmol, 630.2 mg), NMT-TBS (1.2 mmol, 320.0 mg) and TEA (2 mmol, 202.0 mg) were dissolved in dichloromethane, and then HOBT (1.2 mmol, 162.0 mg) and EDC (2 mmol, 383.4 mg) were added and stirred at room temperature overnight. The mixture was washed three times with ammonium chloride, sodium bicarbonate and brine. The organic phases were combined and dried over sodium sulfate. The solvent was removed under reduced pressure. The product was purified by column chromatography (MeOH:DCM=1:20) to obtain a white solid. The yield was 693.0 mg, and the yield was 73%.

[0144] 1H NMR spectrum(400MHz,CDCl3)of T-DCFH-3TBS.1H NMR (400MHz, CDCl3) δ7.25-6.88(m,6H),6.79-6.69(m,3H),6.62(s,3H),5.33-4.99(m,1H),3.9 2-3.85(m,1H),3.20(s,1H),3.00(s,1H),2.85(s,3H),1.07-0.93(m,27H),0.26-0.11(m,18H).

[0145] (5) Compound T-DCFH-TBS (1.0 mmol, 877.4 mg) was dissolved in 10 mL of tetrahydrofuran, and then 1 M tetrabutylammonium fluoride (5 mmol, 5 ml) was slowly added dropwise under nitrogen atmosphere and stirred at room temperature for 2 hours. The mixture was washed three times with NH4Cl and brine, the organic phases were combined, dried over sodium sulfate, and the solvent was removed under reduced pressure. The product was purified by column chromatography (DCM: MeOH = 1:30). The yield was 37.1 mg, and the yield was 70%.

[0146] 1H NMR spectrum(600MHz,DMSO-d6)of T-DCFH.1H NMR (600MHz, DMSO-d6) δ10.47-10.21(m,2H),9.31-9.02(m,1H),7.39-6.59(m,12H),5.07(d,J=76.1Hz ,1H),3.24(s,2H),3.16(s,1H),2.91(t,J=7.0Hz,1H),2.87(s,3H).ESI:536.10[M+H]+,calculatedfor C29H23Cl2NO5:535.10.

[0147] Example 2 Verification of the properties of T-DCFH and its application in protein detection

[0148] 1. Verification of T-DCFH fluorescence properties and determination of excitation and emission wavelengths

[0149] In this example, the fluorescence properties of T-DCFH prepared in Example 1 were verified, and its excitation wavelength and emission wavelength were measured. Fig.16 As shown. The specific operation is as follows: Take the T-DCFH prepared in Example 1, add 2nM HRP and 100μM H2O2 thereto. Then, use relevant instruments to detect its fluorescence properties. The results show that the maximum excitation wavelength of the activated T-DCFH is 513nm, and the maximum emission wavelength is 531nm. It provides important optical parameter basis for subsequent related detection using T-DCFH, ensuring that T-DCFH can be effectively excited to generate fluorescence signals, and the fluorescence signals emitted by it can be accurately detected, thereby improving the accuracy and reliability of detection.

[0150] 2. Verification of T-DCFH for protein sandwich ELISA detection

[0151] Human epididymis protein 4 (HE4) was selected as the detection target, and a series of concentrations of HE4 protein were captured with capture antibodies on commercial ELISA plates. The HRP-labeled antibody was incubated with 250μM T-DCFH and 500μM H2O2. It was recorded by a multifunctional microplate reader that the fluorescence signal showed a good linear relationship in the range of 0 to 2,000pg / mL as the protein concentration increased. In this ELISA reaction, since a large amount of T-DCFH was activated and coupled to the tyrosine in the HE4 protein, the free inactivated T-DCFH was washed away, thereby achieving low-background fluorescence signal amplification for the detection of HE4 protein, laying the foundation for the detection of nucleocapsid protein based on aptamer-activated TSA, as shown in the following example. Fig.17 a- Fig.17 c, where Fig.17 a is a schematic diagram of T-DCFH-based activatable fluorescence amplification for HE4 protein detection in an ELISA plate; Fig.17 b is the fluorescence intensity statistics by adding different concentrations of HE4 protein (including 62.5, 125, 250, 500, 1000 and 2000 pg / mL), showing the fluorescence “TurnOn” based on T-DCFH; Fig.17c is a linear relationship in the range of 0 to 2,000 pg / mL. At the same time, the feasibility and effectiveness of T-DCFH in protein sandwich ELISA detection were demonstrated. Through the synergistic effect with the capture antibody and HRP-labeled antibody, T-DCFH can show a good linear relationship with the increase of protein concentration within a certain concentration range, and can achieve low background fluorescence signal amplification. This provides an experimental basis for the protein detection method based on T-DCFH, and also provides a reference and reference for its application in other protein detection.

[0152] 3. Verification of HRP activation of T-DCFH and exploration of the effect of different HRP concentrations on T-DCFH amplification efficiency

[0153] In 100 μL DPBS containing 250 μM T-DCFH and 500 μM H2O2, 2.0 μg / mL, 1.0 μg / mL, 0.5 μg / mL and 0.25 μg / mL of HRP were added within 200 s, respectively, and then the fluorescence intensity was recorded with a multifunctional microplate reader, and λex=513nm, λem=531nm were selected for detection ( Fig.18 ). The results showed that the fluorescence value was the highest and the amplification efficiency was the strongest when the HRP concentration was 2.0 μg / mL, and there was basically no fluorescence when HRP was absent. In addition, the fluorescence signal increased rapidly and eventually reached the saturation value, and showed good concentration dependence ( Fig.18 ). On the one hand, the correlation between the activation mechanism of T-DCFH and HRP was further verified. Only in the presence of HRP can T-DCFH be effectively activated and produce fluorescence signals, and the intensity of the fluorescence signal shows a good dependence on the concentration of HRP. On the other hand, the optimal HRP concentration was determined to be 2.0μg / mL, which provides a basis for the reasonable selection of HRP concentration based on T-DCFH detection technology, improving detection accuracy and sensitivity, and studying the reaction kinetic mechanism.

[0154] In summary, in this embodiment, the fluorescence properties of T-DCFH itself are first verified to determine its key optical parameters (excitation wavelength and emission wavelength). Then, on this basis, the application effects of T-DCFH in two different protein detection systems (protein sandwich ELISA detection and aptamer-based detection) are explored respectively, including verifying its feasibility, determining related reaction characteristics (such as the relationship between fluorescence signal and protein concentration, the effect of aptamer-coupled HRP concentration on fluorescence signal, etc.), and comprehensively demonstrating the application potential and value of T-DCFH in the field of protein detection, which provides important experimental basis and theoretical basis for the subsequent further development of protein detection technology based on T-DCFH.

[0155] Example 3 Investigating the effect of HRP concentration of different antibody conjugates on T-DCFH amplification efficiency

[0156] Antibody-HRP conjugates are products obtained by coupling different antibodies with HRP. When detecting different types of viral proteins, different specific antibodies need to be coupled with HRP respectively. After these conjugates bind to the corresponding target antigens, they will produce fluorescent signals under specific conditions, thereby realizing the detection of different target antigens. In this embodiment, antibodies that can specifically recognize SARS-CoV-2 nucleocapsid protein are studied to screen out the amplification efficiency of T-DCFH at different HRP concentrations of antibody conjugates when hydrogen peroxide is applied, and to find the HRP concentration conditions of the antibody conjugate that make the amplification efficiency the strongest.

[0157] The experimental operation was as follows: 250 μM T-DCFH and 500 μM H2O2 prepared in Example 1 were added with 2.0 μg / mL, 1.0 μg / mL, 0.5 μg / mL and 0.25 μg / mL of antibody conjugate-HRP, respectively, and then the fluorescence intensity was recorded with a multifunctional microplate reader, and λex=513 nm and λem=531 nm were selected for detection.

[0158] The results are as follows Fig.19 As shown in the figure, it was found that when the concentration of antibody-coupled HRP was 2.0 μg / mL, the fluorescence value was the largest and the amplification efficiency was the strongest. When antibody-coupled HRP was absent, there was basically no fluorescence. In addition, the fluorescence signals increased rapidly, eventually reached the saturation value, and showed good concentration dependence.

[0159] Therefore, it can be seen that in this experiment, the antibody-conjugated HRP concentration has a key influence on the T-DCFH amplification efficiency in the study of antibodies that specifically recognize the SARS-CoV-2 nucleocapsid protein. 2.0 μg / mL is the concentration that maximizes the fluorescence value and has the strongest amplification efficiency. The fluorescence signal increases rapidly to saturation and has a good concentration dependence. This provides a basis for the rational selection of the HRP concentration of the antibody conjugate based on the HRP-T-DCFH technology for detecting the SARS-CoV-2 nucleocapsid protein of the antibody conjugate.

[0160] Example 4 Investigating the effect of HRP (N35-HRP) concentration of nucleic acid aptamer conjugate on T-DCFH amplification efficiency

[0161] In order to detect nucleocapsid protein, HRP is coupled with a nucleic acid aptamer that can specifically recognize nucleocapsid protein to form a nucleic acid aptamer-HRP conjugate. The HRP of different nucleic acid aptamer conjugates is a product obtained by coupling different target molecules or using different nucleic acid aptamers with HRP. This embodiment mainly studies the nucleic acid aptamer conjugate N35-HRP for detecting nucleocapsid protein, aiming to screen out the amplification efficiency of T-DCFH at the HRP concentration of the nucleic acid aptamer conjugate when hydrogen peroxide is applied, and find the HRP concentration condition of the nucleic acid aptamer conjugate N35-HRP that makes the amplification efficiency the strongest.

[0162] The experimental operation is as follows: 20nM, 10nM, 5nM and 1nM of nucleic acid aptamer conjugate N35-HRP were added to 250μM T-DCFH and 500μM H2O2 prepared in Example 1, respectively, and then the fluorescence intensity was recorded with a multifunctional microplate reader, and λex=513nm, λem=531nm were selected for detection.

[0163] The results are as follows Fig. 20 As shown, it was found that when the concentration of aptamer-HRP was 20 nM, the fluorescence value was the largest and the amplification efficiency was the strongest, and there was basically no fluorescence when the aptamer-HRP was absent. In addition, the fluorescence signals increased rapidly, eventually reached the saturation value, and showed good concentration dependence.

[0164] Therefore, it can be seen that in the study of the aptamer conjugate N35-HRP conjugate for detecting nucleocapsid protein in this experiment, the HRP concentration of the aptamer conjugate has an important influence on the amplification efficiency of T-DCFH. 20nM is the concentration that maximizes the fluorescence value and has the strongest amplification efficiency. The fluorescence signal increases rapidly to saturation and has a good concentration dependence. This provides a basis for the rational selection of the HRP concentration of the aptamer conjugate for the HRP-T-DCFH technology for detecting nucleocapsid protein based on the aptamer conjugate.

[0165] Example 5: Verification test that the nucleic acid aptamer of the present invention can activate TSA and significantly improve the sensitivity of nucleocapsid protein detection

[0166] In the above embodiments, the novel fluorescent dye tyramide molecules of the present invention are first synthesized, including the fluorescent dye tyramides shown in formula (I) and formula (II) with specific structures, wherein the synthesis method and steps thereof are described in detail by taking the "Turn On" type fluorescent molecule T-DCFH as an example. Then, the properties of the synthesized T-DCFH are verified, and its excitation wavelength and emission wavelength are determined, and the feasibility and effectiveness of T-DCFH in protein detection are further confirmed in the protein sandwich ELISA detection and HRP-labeled aptamer activation verification. At the same time, the effects of different HRP concentrations, HRP concentrations of different antibody conjugates, and HRP concentrations of different aptamer conjugates on the amplification efficiency of T-DCFH are also discussed respectively. The optimal HRP concentration, the HRP concentrations of different antibody conjugates, and the HRP concentrations of different aptamer conjugates are confirmed.

[0167] Therefore, in this example, a comprehensive and in-depth validation test was conducted on the sensitivity of the activatable TSA of nucleic acid aptamers in detecting nucleocapsid protein. (In the data analysis, F0 represents the fluorescence intensity without the target protein. All data are mean ± standard deviation, n = 3).

[0168] The experimental procedure was as follows: First, three different formulations were constructed, including formulation 1: triple FITC-labeled aptamers ( Fig.21 a); Formulation 2: Triple aptamer-HRP with “Always on” FT ( Fig.21 b); Formulation 3: Triple aptamer-HRP with "Turn on" T-DCFH ( Fig.21 c; the best formula of the present invention), and was modified on the surface of microscale beads by capture antibodies to capture nucleocapsid protein. And three formulas with different concentrations of SARSCoV-2 nucleocapsid protein ranging from 0, 5, 10, 50, 100, 200, 400, 800 to 1000 ng / mL were analyzed by flow cytometry, specifically: In formula 1, the nucleic acid aptamer was labeled with a fluorescent dye (FITC) and can be directly read by a flow cytometer, such as Fig.21 d. In formula 2, FT was added to amplify the signal under the catalysis of HRP-labeled aptamer by commercial “ALWAYS ON” FT, as shown in Fig.21 e. In formula 3, after the HRP-labeled aptamer targets the nucleocapsid protein, the “Turn on” molecule T-DCFH is used to amplify the fluorescence signal, as shown in Fig.21 f.

[0169] In order to highlight the effect of the "aptamer-HRP / T-DCFH" (Formula 3) of the present invention, "aptamer-FITC" (Formula 1) and "aptamer-HRP / FT" (Formula 2) were introduced as controls in this example and modified on the surface of microscale beads by capture antibodies to capture nucleocapsid proteins.

[0170] The flow cytometry results showed that almost no fluorescence shift was observed in formula 1. Although there was a significant fluorescence shift in formula 2, it is worth noting that the background fluorescence was higher in the absence of nucleocapsid protein. In formula 3, not only was a significant fluorescence shift observed, but the fluorescence background was also lower, which was consistent with the results of quantitative fluorescence analysis. The significant fluorescence shift can indicate a higher sensitivity for nucleocapsid protein detection, while the lower fluorescence background can significantly improve the accuracy of detection. In addition, from Fig.21 g, formula 3 has a higher fluorescence intensity. At the same time, the signal-to-noise ratio (SNR) analysis shows that the SNR of formula 3 is higher than that of formula 1 and formula 2 ( Fig.21 h), which also shows that formula 3 has higher accuracy and sensitivity, and the detection results are more reliable.

[0171] The above results show that this example fully proves that the triple aptamer-HRP with "Turn on" T-DCFH (optimal formula 3 of the present invention) has significant advantages in detecting nucleocapsid protein by analyzing the sensitivity of three different formulas for detecting nucleocapsid protein. It not only has high detection sensitivity and can accurately detect in low-concentration nucleocapsid protein samples, but also has low fluorescence background, which improves the accuracy and reliability of detection. This provides a new and efficient method for the detection of nucleocapsid protein, which is expected to play an important role in clinical diagnosis and scientific research.

[0172] Example 6: Verification test that the nucleic acid aptamer of the present invention can activate TSA and significantly improve the specificity of nucleocapsid protein detection

[0173] In this example, in order to further evaluate the specificity of the activatable TSA of the nucleic acid aptamer of the present invention to detect the nucleocapsid protein, the nucleocapsid proteins of four infectious viruses, influenza A virus H3N2, HCOv-Hku1, Hcov-229E and MERS-Cov, were selected for comparative experiments with the SARS-CoV-2 nucleocapsid protein of the present invention.

[0174] The experimental process is as follows: 1,000 ng / mL of different nucleocapsid proteins were incubated with antibody-modified beads for 2 hours, followed by the addition of 200 nM HRP-labeled aptamers (N35, N10, and N2-62), and incubated at room temperature for 1 hour, followed by washing 3 times (directly adding the three aptamers to the reaction system at the same time (triple aptamers) and binding to the nucleocapsid proteins of different viruses). After that, 250 μM T-DCFH and 500 μM H2O2 were added, incubated with the aptamer sandwich structure for 0.5 hours, and then washed 3 times before fluorescence analysis on CytoFLEXLX (Beckman Coulter, USA). In this embodiment, the aptamers and their sequences are shown in Table 1.

[0175] Table 1. Three nucleic acid aptamers and their sequences in the present invention

[0176] name Number of bases (nt) 5'～3' N35 -- SEQ ID NO.1 N10 -- SEQ ID NO.2 N2-62 -- SEQ ID NO.3

[0177] Note: All FITC-labeled or HRP-labeled aptamers are labeled at the 5' end of the sequence.

[0178] The synthesis and native-PAGE purification of the three nucleic acid aptamer HRP conjugates are as follows: Fig. 22 a. Fig. 22 b): 5'-amino-modified DNA is reacted with the heterobifunctional linker SMCC to convert into thiol-reactive maleimide. At the same time, the primary amino group of HRP is modified with SATA to generate (protected) thiol-activated SATA-HRP. After deprotection of SATA-HRP to thiolate HRP with hydroxylamine, it reacts with maleimide-activated oligonucleotide to form a stable thiol ester bond between DNA and HRP. Finally, the HRP-oligonucleotide conjugate is separated from the unreacted oligonucleotide and HRP by native-PAGE purification.

[0179] Prepare 12% native polyacrylamide gel, mix 10μL DNA (2μM) with 2μL 6x loading buffer, run at 80V for 10min in 1×TBE buffer (89mmol / L Tris-boric; 2mmol / L EDTA; pH=8.2-8.4), and then run at 110V for 60min. After staining with 1x Gel-Red (Everbright, Suzhou, USA, catalog number: S2001) for 15min, the gel was imaged using the ChemiDoc XRS+ system (Bio-Rad, USA). 25-500bp DNAMarker (Shanghai Shenggong Biotechnology Co., Ltd., catalog number: B600303) was used as a reference. Finally, the three aptamer-HRP conjugates were analyzed by MALDI-TOF mass spectrometry.

[0180] Flow cytometry results showed that the deviation of influenza A H3N2, HCOv-Hku1, Hcov-229E and MERS-Cov proteins was small, while the deviation of SARS-CoV-2 nucleocapsid protein was obvious ( Fig.23 a) From the corresponding Fig. 22 The fluorescence intensity statistics of a Fig.23 b), the value of SARS-CoV-2 nucleocapsid protein is higher than that of the other four virus-related nucleocapsid proteins, which indicates that formula 3 has higher specificity. In addition, the signal-to-noise ratio quantitative analysis ( Fig.23 c) showed that the signal-to-noise ratio of the SARS-CoV-2 group was about 7, which was significantly higher than that of the other four groups. F0 represents the fluorescence intensity without the target protein, and FBeads represents the fluorescence intensity of the beads alone. All data are mean ± standard deviation, n = 3. To further confirm the fluorescence enhancement effect of formula 3, confocal laser microscopy imaging was performed. Imaging results ( Fig.23 d) shows that the fluorescence signal of formula 3 was significantly enhanced when incubated with 1000ng / mL SARS-CoV-2 nucleocapsid protein compared with the other two groups. The scale bar is 5μm. Fig.23 d Quantitative analysis of the corresponding slices in the image (white arrows) ( Fig.23 e), the results are consistent with the imaging results.

[0181] The above results show that the detection method of the nucleic acid aptamer-activated TSA of the present invention has high specificity for SARS-CoV-2 nucleocapsid protein and can effectively distinguish SARS-CoV-2 nucleocapsid protein from the nucleocapsid proteins of the other four infectious viruses.

[0182] Example 7 Comparative test of the detection effect of tyramide, a fluorescent dye with different structures, on SARS-CoV-2 nucleocapsid protein

[0183] In this example, in order to clarify the detection effect of fluorescent dye tyramide with different structures on SARS-CoV-2 nucleocapsid protein, a comparative test was conducted on the amplification effect, sensitivity, specificity and accuracy of fluorescent dye tyramide with different structures in the detection of SARS-CoV-2 nucleocapsid protein in Example 1.

[0184] The experimental process is as follows:

[0185] 1. According to the best synthesis method in Example 1, the fluorescent dye tyramines shown in formula (I-1) to formula (I-6) and formula (II-1) were synthesized respectively. And for seven fluorescent dye tyramines with different structures, three formulas similar to those in Example 6 were constructed respectively: Formula A: triple FITC-labeled aptamer (as a control); Formula B: triple aptamer-HRP with "Always on" FT; Formula C: triple aptamer-HRP with corresponding fluorescent dye tyramine structure (such as formula (I-1) corresponding to "Turn on" formula (I-1)-T-DCFH). And it was modified on the surface of microscale beads by capture antibodies to capture nucleocapsid protein.

[0186] 2. Detection of different concentrations of nucleocapsid protein

[0187] Three formulations with different concentrations of SARS-CoV-2 nucleocapsid protein ranging from 0, 5, 10, 50, 100, 200, 400, 800 to 1000 ng / mL were analyzed by flow cytometry as follows:

[0188] In formulation A, the aptamer is labeled with a fluorescent dye (FITC) and can be directly read by flow cytometry.

[0189] In formulation B, FT was added to amplify the signal under the catalysis of HRP-labeled aptamer by commercial “ALWAYS ON” FT.

[0190] In formulation C, after the HRP-labeled aptamer targets the nucleocapsid protein, the fluorescence signal is amplified using a "Turn on" molecule of the corresponding structure.

[0191] The fluorescence shift, signal-to-noise ratio, and fluorescence background of tyramide, a fluorescent dye with different structures, were statistically analyzed. The specific results are shown in Tables 2 to 4.

[0192] Table 2. Fluorescence shift of tyramide fluorescent dyes with different structures

[0193]

[0194]

[0195] Table 3. Signal-to-noise ratio of tyramide fluorescent dyes with different structures

[0196] Fluorescent dye tyramide structure Formulation A signal-to-noise ratio Formulation B signal-to-noise ratio Formulation C signal-to-noise ratio Formula (I-1) 3 4 9 Formula (I-2) 2 3 8 Formula (I-3) 2 3 8 Formula (I-4) 2 2 8 Formula (I-5) 1 2 7 Formula (I-6) 1 2 7 Formula (II-1) 3 3 9

[0197] Table 4. Fluorescence background of tyramide fluorescent dyes with different structures

[0198] Fluorescent dye tyramide structure Formula A Fluorescent Background Formula B Fluorescent Background Formula C Fluorescent Background Formula (I-1) Low (no signal amplification) High background in the absence of protein Low Formula (I-2) Low (no signal amplification) High background in the absence of protein Low Formula (I-3) Low (no signal amplification) High background in the absence of protein Low Formula (I-4) Low (no signal amplification) High background in the absence of protein Low Formula (I-5) Low (no signal amplification) High background in the absence of protein Lower Formula (I-6) Low (no signal amplification) High background in the absence of protein Lower Formula (II-1) Low (no signal amplification) High background in the absence of protein Low

[0199] From the above experimental results, it can be seen that fluorescent dye tyramide with different structures exhibits different characteristics in the detection of SARS-CoV-2 nucleocapsid protein:

[0200] (1) Amplification effect and sensitivity: All fluorescent dye tyramides of the structure in formula C show more obvious fluorescence shift than formula A, indicating that they are superior to formula A (FITC labeled only) in terms of signal amplification and detection sensitivity. Among them, formula (I-1), (I-2), (I-3), (I-4) and (II-1) have the most obvious fluorescence shift in formula C, which may have better signal amplification effect and detection sensitivity; while formula (I-5) and formula (I-6) have slightly smaller fluorescence shifts, but still have obvious shifts compared to formula A, and the fluorescence background is moderate, so they also have good signal amplification effect and detection sensitivity. This may be because the fluorescent dye tyramide of the structural type of formula (I) has a specific combination of substituents and spatial structure, which enables it to play a more effective role in binding to the nucleocapsid protein and in the signal amplification process. The fluorescent dye tyramide of the structural type of formula (II) also has a similar chemical structure and active site, which enables it to participate in the signal amplification process better, so both show good signal amplification and detection sensitivity characteristics.

[0201] At the same time, among the fluorescent dyes of the structural type of formula (I), since the carboxyl-containing aromatic rings of formula (I-5) and formula (I-6) have electron-withdrawing substituents (nitro, 1,2,3,4-tetrachloro), their binding efficiency with the nucleocapsid protein or the degree of signal amplification may be affected, and therefore the results are slightly inferior to those of formula (I-1), (I-2), formula (I-3), and formula (I-4).

[0202] (2) Specificity and accuracy: The fluorescent dye tyramide in formula C can be clearly distinguished from formula A (almost no deviation) in the presence of nucleocapsid protein, and in the absence of nucleocapsid protein, the background fluorescence of most structures of fluorescent dye tyramide (Formula (I-1), Formula (I-2), Formula (I-3), Formula (I-6) and Formula (II-1), etc.) in formula C is very low. Combined with the signal-to-noise ratio analysis, the fluorescent dye tyramide of these structures performs better in terms of specificity and accuracy; and although the fluorescence background of Formula (I-4) and Formula (I-5) is slightly higher than that of other structures, it is still significantly lower than that of Formula A, and therefore also has good specificity and accuracy.

[0203] In summary, the fluorescent dye tyramine with structures such as formula (I-1), formula (I-2), formula (I-3), formula (I-6) and formula (II-1) has the best comprehensive performance in the detection of nucleocapsid protein, including signal amplification effect, sensitivity, specificity and accuracy; at the same time, the fluorescent dye tyramine with structures of formula (I-4) and formula (I-5) has good comprehensive performance in the detection of nucleocapsid protein. In practical applications, fluorescent dye tyramine with a suitable structure can be selected for detection according to specific needs.

[0204] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An activatable signal amplification system based on the fluorescent dye tyramide, characterized in that: The activatable signal amplification system includes a fluorescent dye tyramide, and the fluorescence of the fluorescent dye tyramide can be activated only in the presence of ROS, thereby realizing activated tyramide signal amplification.

2. The activatable signal amplification system according to claim 1, characterized in that: The fluorescent dye tyramine comprises a structural formula as shown in formula (I) or formula (II): Among them, R1 or R2 includes any one or more of hydrogen atoms, fluorine atoms, chlorine atoms, bromine atoms or iodine atoms; R3, R5 or R6 includes any one or more of hydrogen atoms or chlorine atoms; R4 includes any one or more of hydrogen atoms, chlorine atoms or nitro groups.

3. The activatable signal amplification system according to claim 2, characterized in that: The structural formula represented by the formula (I) includes any one of formula (I-1) to formula (I-6):

4. The activatable signal amplification system according to claim 2, characterized in that: The structural formula represented by the formula (II) includes formula (II-1):

5. A biomolecule based on an activatable signal amplification strategy, characterized in that: The biological molecule comprises the fluorescent dye tyramide, and the fluorescent dye tyramide comprises the fluorescent dye tyramide in any one of the activatable signal amplification systems of claims 1-4.

6. The biomolecule according to claim 5, characterized in that The biomolecules also include any one or more combinations of antibodies, nucleic acid aptamers, polypeptides, and small molecules that specifically recognize antigens.

7. The biomolecule according to claim 6, characterized in that Horseradish peroxidase is used to label nucleic acid aptamers, wherein the nucleic acid aptamers are nucleic acid sequences that can specifically recognize antigens, and the nucleic acid sequences of the nucleic acid aptamers are as shown in SEQ ID NO.1, SEQ ID NO. 2 and SEQ ID NO.

3.

8. An activatable signal amplification detection method based on the fluorescent dye tyramide, characterized in that: The method activates the fluorescent dye tyramide by using the biomolecule according to any one of claims 5 to 7, thereby specifically identifying the antigen to be detected and achieving signal amplification.

9. The activatable signal amplification detection method according to claim 8, characterized in that: The detection method comprises the following steps: (1) incubating the antigen to be detected and the antibody-modified beads, and then co-incubating with the HRP-labeled aptamer to form a recognition molecule sandwich structure for detecting the antigen to be detected; (2) Fluorescent dyes tyramide and H2O2 are then added to achieve enrichment, thereby specifically identifying the antigen to be tested and achieving signal amplification.

10. A use of fluorescent dye tyramine for preparing a preparation for improving the accuracy, sensitivity and specificity of antigen detection, characterized in that: The fluorescent dye tyramide includes the fluorescent dye tyramide in any one of claims 1 to 4 that can be activated in the signal amplification system.

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