HBV PreS1 Antibody Quantitative Detection Test Strip Based on AIE Molecule, Its Preparation Method and Application

Abstract

The application discloses an AIE molecule-based HBV PreS1 antibody quantitative detection test strip as well as a preparation method and application thereof, and relates to the fields of molecular biology, immunology and biological detection technology.The test strip comprises a sample pad, a mark pad, a nitrocellulose membrane (NC membrane), a water absorption pad and a bottom plate, the NC membrane detection line is coated with an HBV PreS1 antigen and is used for capturing PreS1 antibodies in a sample, and the mark pad is fixed with Anti-Human IgG antibodies coupled with AIE molecules and is used for combining with the PreS1 antibodies in the sample to form an immune complex.The test strip can realize rapid detection of the PreS1 antibodies, realize a fluorescence enhancement effect of a detection result, and, in combination with a fluorescence reading instrument or a fluorescence reading card equipment, the detection result can be subjected to digital quantitative analysis, is simple and convenient to operate, rapid to detect, significantly improves detection sensitivity and realizes quantitative analysis.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology, immunology, and biodetection technology, specifically to a quantitative test strip for HBV PreS1 antibody based on AIE molecules, its preparation method, and its application. Background Technology

[0002] Hepatitis B virus (HBV) infection remains a serious global public health problem, potentially progressing to chronic hepatitis, cirrhosis, and even hepatocellular carcinoma (HCC). During HBV infection, the body generates an immune response against different viral structural proteins. The PreS1 region, located in the PreS region of the HBV surface antigen, is a crucial domain for viral binding to host hepatocyte receptors and plays a key role in viral invasion. PreS1 antibodies against the PreS1 antigen are of significant clinical importance in HBV infection diagnosis, immune response assessment, and vaccine efficacy monitoring. Therefore, establishing a sensitive, rapid, and accurate PreS1 antibody detection method is of great value for HBV infection screening, treatment monitoring, and prognostic assessment.

[0003] Currently, the main methods for detecting HBV-related antibodies include enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and colloidal gold immunochromatography. ELISA and chemiluminescence immunoassay offer high sensitivity and accuracy, but they typically require specialized equipment and technicians, involve complex procedures, and are time-consuming. They are mostly conducted in laboratories or large medical institutions, making them unsuitable for primary healthcare facilities and point-of-care testing (POCT) applications. Colloidal gold immunochromatography, due to its simplicity, rapid detection, and lack of complex instruments, has been widely used for the rapid detection of various infectious diseases.

[0004] However, traditional colloidal gold immunochromatographic test strips rely primarily on the visible color signal generated by colloidal gold particles for visual interpretation. Their detection sensitivity is limited, with the detection limit failing to reach the ng / mL level, and they cannot accurately distinguish concentration gradients, making it difficult to achieve highly sensitive detection of low concentrations of target molecules. Furthermore, this type of method struggles to provide precise quantitative analysis of the results, and weak signals or even false negatives are common in samples with low viral loads or low antibody levels, limiting its application in highly sensitive and precise quantitative detection.

[0005] Therefore, developing a novel detection technology that combines the advantages of easy operation and rapid detection of immunochromatographic test strips with significantly improved detection sensitivity and quantitative analysis is of great significance for enhancing the accuracy and application scope of HBV-related antibody detection. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides an HBV PreS1 antibody quantitative detection test strip based on AIE molecules, its preparation method, and its application. This overcomes the limitations of traditional colloidal gold test strips in terms of sensitivity and accuracy, meeting the needs of clinical point-of-care testing and rapid on-site detection.

[0007] According to one aspect of the present invention, a method for preparing an HBV PreS1 antibody quantitative detection test strip based on AIE molecules is provided, comprising:

[0008] S1, Synthesize the AIE fluorescent molecule DPTPA-CHO;

[0009] Add 4-diphenylaminobenzaldehyde to chloroform, then add N-bromosuccinimide in portions, and stir overnight at room temperature;

[0010] Then, pure water was added and mixed well. Dichloromethane was added to separate the organic phase. The organic phase was washed with saturated sodium chloride solution, dehydrated with sodium sulfate, filtered, and dried by rotary evaporation. The intermediate product 4-[bis(4-bromophenyl)amino]benzaldehyde was obtained by purification with n-hexane as the eluent.

[0011] 4-[bis(4-bromophenyl)amino]benzaldehyde and 4-pyridylboronic acid were reacted in a mixed solvent of water, ethanol and toluene under the catalysis of tetra(triphenylphosphine)palladium and the protection of an inert gas. Cs2CO3 was added to the system. After the reaction was completed, 4-(bis(4-(pyridin-4-yl)phenyl)amino)benzaldehyde was obtained, denoted as DPTPA-CHO.

[0012] S2, AIE fluorescent molecule DPTPA-CHO conjugated with Anti-Human IgG antibody;

[0013] The DPTPA-CHO and DSPE-PEG-NHS obtained in S1 were dissolved in a mixed solution of tetrahydrofuran and water, sonicated and stirred overnight, and then filtered through a filter membrane to obtain DPTPA-NPs.

[0014] Add DPTPA-NPs and Anti-Human IgG to PBS buffer, react at room temperature, then quench with Tris buffer, and dialyze to remove free DPTPA-NPs to obtain the AIE fluorescent molecule DPTPA-CHO and Anti-Human IgG antibody conjugate product DPTPA-NPs@Anti-Human IgG.

[0015] S3, Prepare the PreS1 antibody fluorescent test strip with AIE-enhanced luminescence;

[0016] The detection lines on the NC membrane were sprayed with HBV PreS1 antigen solution, and the control lines were sprayed with HB2 antibody. The sprayed NC membrane was then dried in an oven, then immersed in BSA solution for sealing, and finally dried at room temperature and stored in a sealed, light-protected container.

[0017] DPTPA-NPs@Anti-Human IgG were sprayed onto a glass cellulose membrane, then dried at room temperature and stored in a sealed container away from light.

[0018] The NC membrane, glass cellulose membrane, sample pad, and absorbent pad are attached to the backing plate in sequence. The assembled backing plate with each component attached is then cut using a strip cutter. Finally, the strip is placed into the plastic housing of the test strip to complete the assembly.

[0019] Optionally, in S1, the molar ratio of 4-diphenylaminobenzaldehyde to N-bromosuccinimide is 1:2.1; the volume ratio of the eluent n-hexane to dichloromethane is 4:5; and the molar ratio of 4-[bis(4-bromophenyl)amino]benzaldehyde to 4-pyridylboronic acid is 33:80.

[0020] Optionally, in S1, the inert gas is argon, the volume ratio of water:ethanol:toluene in the mixed solvent is 1:1:2, and the reaction condition is stirring overnight at 100 °C.

[0021] Optionally, in S2, the mass ratio of DPTPA-CHO to DSPE-PEG-NHS is 1:5; and the molar ratio of DPTPA-NPs to Anti-Human IgG is 5:1.

[0022] Optionally, in S2, during the reaction of DPTPA-NPs with Anti-Human IgG, the pH of the solution is adjusted to 8 using sodium bicarbonate.

[0023] Optionally, in S2, the ultrasonic treatment time is not less than 1 min; the filter membrane pore size is 0.22 µm; and the reaction time of DPTPA-NPs and Anti-Human IgG at room temperature is not less than 2 h.

[0024] Optionally, in S3, the concentration of the HBV PreS1 antigen solution used is 1.5–2 mg / mL; the spraying amount of DPTPA-NPs@Anti-Human IgG is 3–4 µL / cm. 2 .

[0025] Optionally, in S3, the NC membrane is sealed by immersing it in a 2% w / v BSA solution.

[0026] According to another aspect of the present invention, an HBV PreS1 antibody quantitative detection strip based on AIE molecules is provided, which is prepared by the preparation method of the HBV PreS1 antibody quantitative detection strip based on AIE molecules as described above.

[0027] According to another aspect of the present invention, the application of the HBV PreS1 antibody quantitative test strip based on the AIE molecule as described above in the preparation of a hepatitis B virus PreS1 antibody detection kit is provided.

[0028] The beneficial effects of this invention are:

[0029] The test strip of this invention uses an HBV PreS1 antigen-coated NC membrane to capture PreS1 antibodies in the sample. Anti-Human IgG, as a secondary antibody, can bind to the PreS1 antibody to form an "antigen-antibody-secondary antibody" complex structure. AIE molecules, as signal molecules, are coupled with Anti-Human IgG, and a fluorescent signal can be observed under ultraviolet light. The fluorescence signal amplification mechanism can significantly improve the detection sensitivity. At the same time, the fluorescence card reader can realize the quantitative reading and digital quantitative output of the test results. It not only retains the advantages of colloidal gold test strips such as "rapid detection in 15 minutes" and "no professional training required", but also overcomes the problems of insufficient sensitivity and accuracy through the AIE fluorescence signal amplification mechanism. It provides accurate data for low viral load samples and dynamic monitoring of antibody titers, and provides a universal and innovative solution for the detection of hepatitis B virus biomarkers. Attached Figure Description

[0030] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0031] Figure 1 The diagram shows the detection principle of the PreS1 antibody fluorescent test strip with enhanced AIE luminescence, (a) a schematic diagram of the composition of the PreS1 antibody fluorescent test strip with enhanced AIE luminescence, (b) a schematic diagram of the test results for negative samples, and (c) a schematic diagram of the test results for positive samples.

[0032] Figure 2 A route diagram for the synthesis of DPTPA-CHO;

[0033] Figure 3 The mass spectrum of the intermediate product 4-[bis(4-bromophenyl)amino]benzaldehyde;

[0034] Figure 4 Characterization of DPTPA-CHO: (a) UV absorption and emission analysis, (b) mass spectrum;

[0035] Figure 5 The synthetic route diagram for DPTPA-NPs@Anti-Human IgG;

[0036] Figure 6 Characterization diagrams of DPTPA-NPs: (a) UV absorption and emission analysis, (b) particle size distribution, (c) mass spectrum, (d) TEM characterization;

[0037] Figure 7 The stability test results for DPTPA-NPs are shown in (a) UV absorption analysis and (b) emission analysis.

[0038] Figure 8 Characterization diagrams of DPTPA-NPs@Anti-Human IgG: (a) pH optimization, (b) screening of nanoparticle-antibody conjugation ratio, (c) changes in particle size and zeta potential of DPTPA-NPs before and after conjugation, and (d) UV absorption analysis of DPTPA-NPs@Anti-Human IgG.

[0039] Figure 9 Screening chart for coating antigen concentration;

[0040] Figure 10 Screening diagram for optimal spraying amount of DPTPA-NPs@Anti-Human IgG;

[0041] Figure 11 Immunoreactivity kinetics analysis of PreS1 antibody fluorescent test strips for AIE enhanced luminescence;

[0042] Figure 12 The standard curve of the PreS1 antibody fluorescent test strip for AIE enhanced luminescence: (a) visual interpretation of the result, (b) quantitative detection result of the instrument;

[0043] Figure 13 For the specificity evaluation chart, (a) visual interpretation results, (b) instrument quantitative detection results;

[0044] Figure 14 For repeatability evaluation charts, (a) visual interpretation results and (b) quantitative detection results;

[0045] Figure 15 The thermal stability evaluation diagram shows (a) visual interpretation results and (b) quantitative instrument detection results.

[0046] Figure 16 The transportation stability evaluation diagram includes (a) visual interpretation results and (b) quantitative instrument detection results.

[0047] Figure 17 Comparative detection images of PreS1 antibody fluorescent test strips for AIE enhanced luminescence. Detailed Implementation

[0048] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, and not all of them. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present application. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present application can be combined with each other.

[0049] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0050] Unless otherwise specified, all conditions in the examples were performed under standard conditions or according to the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are commercially available standard products. Unless otherwise stated, all technical and scientific terms herein have the meanings commonly understood by one of ordinary skill in the art.

[0051] Aggregation-induced emission (AIE) materials have attracted widespread attention in fields such as biosensing, fluorescence imaging, and sensing analysis because they can generate significantly enhanced fluorescence signals in the aggregated state and have good photostability and resistance to photobleaching.

[0052] This invention proposes a fluorescence quantitative test strip based on aggregation-induced emission (AIE) molecular technology. By introducing AIE molecules, in a solid aggregated state, AIE molecules have significant fluorescence enhancement effects and anti-photobleaching properties. Combined with a fluorescence reader or fluorescence card reader, it can realize digital quantitative analysis of test results, achieving ultra-high sensitivity detection at the ng / mL level, and supporting digital quantitative output, thereby overcoming the shortcomings of insufficient sensitivity and difficulty in quantification of traditional colloidal gold test strips.

[0053] like Figure 1 The test strip of the present invention uses HBV PreS1 antigen coated NC membrane to capture PreS1 antibody in sample. Anti-Human IgG as secondary antibody binds to PreS1 antibody and forms an "antigen-antibody-secondary antibody" complex structure after coupling. AIE molecule as signal molecule, fluorescent signal can be observed under ultraviolet light, and quantitative reading can be achieved by fluorescent card reader.

[0054] The main instruments and consumables involved include:

[0055] Fluorescent test strip reader (model: JY96-ⅡN), FLS-1000 photoluminescence spectrometer (model: FS5-S-1067-0715-A2), transmission electron microscope (model: Aztec X-Max 80T). 10g 4-diphenylaminobenzaldehyde (Anhydrous acetonitrile), ≥99.9% analytical grade 250 mg tetrakis(triphenylphosphine)palladium(0), ≥99.9% analytical grade 25 g N-bromosuccinimide (NBS), ≥99.9% analytical grade sodium sulfate, ≥99.9% analytical grade dichloromethane.

[0056] The technical solution and its effects of the present invention will be fully explained below through specific implementation steps and performance analysis.

[0057] Example 1: Synthesis and Characterization Analysis of DPTPA-CHO:

[0058] (1) Synthesis of DPTPA-CHO:

[0059] The synthesis method of DPTPA-CHO is as follows: Figure 2 As shown, 4-diphenylaminobenzaldehyde was added to 20 mL of chloroform, and NBS (N-Bromosuccinimide) was added slowly in two portions. The molar ratio of 4-diphenylaminobenzaldehyde to NBS was 1:2.1. The mixture was stirred overnight at room temperature.

[0060] Add pure water to the reaction flask, mix well, then add dichloromethane (DCM) to separate the organic phase. Wash the organic phase three times with saturated sodium chloride solution, remove water with sodium sulfate, filter, and evaporate to dryness. The eluent ratio of n-hexane to DCM is 4:5 (volume ratio). The purified product yields the intermediate 4-[bis(4-bromophenyl)amino]benzaldehyde.

[0061] 0.33 mmol of 4-[bis(4-bromophenyl)amino]benzaldehyde and 0.8 mmol of 4-pyridylboronic acid reacted with tetrakis(triphenylphosphine)palladium catalyst to give 4-(bis(4-(pyridin-4-yl)phenyl)amino)benzaldehyde (DPTPA-CHO), wherein 0.66 mmol of Cs₂CO₃ (cesium carbonate) was added to the system. The reaction conditions were: Ar (argon) atmosphere at 100 °C with stirring overnight. The reaction solvent was water:ethanol:toluene = 1:1:2, i.e., 1.6 mL:1.6 mL:3.2 mL.

[0062] (2) Characterization analysis of DPTPA-CHO

[0063] DPTPA-CHO was synthesized in two steps. The mass spectrum of the intermediate product 4-[bis(4-bromophenyl)amino]benzaldehyde is shown below. Figure 3 As shown, the molecular weight is 431.9460, which is consistent with the theoretical calculation.

[0064] The UV absorption and fluorescence emission spectra of DPTPA-CHO are as follows: Figure 4 As shown in (a), it exhibits maximum absorption at 360 nm and emits light at a wavelength of 510 nm. The mass spectrum is shown in 4(b), with a molecular weight of 428.1798, which is consistent with the theoretical calculation, indicating that DPTPA-CHO has been successfully synthesized and can be used for the next step of nanoparticle preparation.

[0065] Example 2: Preparation and performance testing of DPTPA-NPs@Anti-Human IgG:

[0066] (1) Preparation of DPTPA-NPs@Anti-Human IgG:

[0067] DPTPA-CHO and DSPE-PEG-NHS (distearylphosphatidylethanolamine-polyethylene glycol-active ester) were dissolved in a mixed solution of tetrahydrofuran and water at a mass ratio of 1:5. The solution was sonicated for 2 min and stirred overnight. The mixture was then filtered through a 0.22 µm filter membrane to complete the preparation of DPTPA-NPs.

[0068] like Figure 5 Antibody conjugation was achieved by reacting the NHS on DPTPA-NPs with the NH2 on the antibody. DPTPA-NPs and Anti-Human IgG were added to PBS and reacted at room temperature for 2 hours. The reaction was then quenched with Tris buffer, and free DPTPA-NPs were removed by dialysis, yielding the AIE fluorescent molecule DPTPA-CHO and Anti-Human IgG antibody-conjugated product, namely DPTPA-NPs@Anti-Human IgG.

[0069] (2) Performance testing of DPTPA-NPs@Anti-Human IgG:

[0070] DPTPA-CHO has a very small molecular weight and can penetrate ultrafiltration tubes. After forming nanoparticles (DPTPA-NPs), after ultrafiltration and centrifugation, the DPTPA-NPs cannot penetrate the ultrafiltration membrane and remain in the upper layer of the ultrafiltration tube. The synthesis of DPTPA-NPs is then successful. Figure 6 As shown in (a), a clear red shift is observed between the absorption and emission peaks, with the maximum UV absorption increasing from 360 nm to 410 nm and the maximum emission increasing from 510 nm to 582 nm. The particle size distribution is as follows: Figure 6 As shown in (b), the particles exhibit a normal distribution and a narrow particle size distribution range, demonstrating that the prepared DPTPA-NPs have relatively uniform particle size. The mass spectrum is shown below. Figure 6 (c) It exhibits a normal distribution trend, which is basically consistent with the particle size distribution. The particle morphology under transmission electron microscopy (TEM) is as follows: Figure 6 As shown in (d), the particle size is around 65 nm.

[0071] The synthesized DPTPA-NPs were continuously irradiated with strong light for 15 minutes, and the absorption and emission were measured every minute. The results are as follows: Figure 7 As shown, DPTPA-NPs showed almost no change in absorption and emission after 15 minutes of continuous irradiation, indicating good photostability.

[0072] DSPE-PEG-NHS is anchored to the surface of nanoparticles through hydrophobic interactions. The hydrophilic PEG chains extend outwards to form a steric hindrance layer, while the terminal NHS groups are exposed to the aqueous phase. The NHS groups react with free amino groups on the antibody surface to form covalent amide bonds, thus immobilizing the antibody on the nanoparticle surface. pH is a key factor affecting the coupling of Anti-Human IgG and DPTPA-NPs; therefore, the pH of the reaction system was investigated, and the results are as follows: Figure 8 As shown in (a), the reaction occurs more readily in a weakly alkaline environment, with the highest coupling efficiency at pH 8. Figure 8 As shown in (b), the T-line fluorescence signal was strongest at pH 8 and a DPTPA-NPs to antibody ratio of 5:1. After DPTPA-NPs-conjugated antibody was used to synthesize DPTPA-NPs@Anti-Human IgG, as shown in [the image]... Figure 8 As shown in (c), the Zeta potential changed from -3.17 mV to -15.23 mV, the hydration particle size increased from 65 nm to 86.17 nm, and the absorption and emission of DPTPA-NPs@Anti-Human IgG also showed a red shift, as... Figure 8As shown in (d), there is a maximum absorption peak at 416 nm, indicating that DPTPA-NPs@Anti-Human IgG was successfully prepared.

[0073] Example 3: Construction and performance analysis of AIE-enhanced PreS1 antibody fluorescent test strip:

[0074] (1) Screening by coating antigen concentration:

[0075] Different concentrations of antigen solution (0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, and 2.5 mg / mL) were used to coat the NC membrane. The optimal coating antigen concentration was selected by visually observing the T-line color development effect. Results are as follows... Figure 9 The optimal coating antigen concentration is 2 mg / mL.

[0076] (2) Optimal spraying amount of DPTPA-NPs@Anti-Human IgG:

[0077] The optimal spraying amounts of DPTPA-NPs@Anti-Human IgG were set at 1, 2, 3, 4, and 5 µL / cm. 2 The group with the most significant difference between positive and negative samples was selected as the final coating amount standard. Figure 10 It can be determined that the spraying amount is 4 µL / cm 2 At this time, the positive to negative sample test ratio (P / N) is the largest. If the amount of DPTPA-NPs@Anti-Human IgG sprayed is too high, it will cause the C line to be blurred and false positives will occur.

[0078] (3) Assembly of AIE-enhanced PreS1 antibody fluorescent test strips:

[0079] Spray the test line onto the NC membrane with the HBV PreS1 antigen solution at the optimal concentration (preferably 2 mg / mL), and spray the control line with HB2 antibody (HBV PreS1 antibody reference). (In this embodiment, the amount of HB2 antibody sprayed is not optimized; conventional spraying, such as 4 µL / cm, is sufficient.) 2 After spraying, the NC film is placed in an oven to dry, then immersed in a 2% w / v BSA solution to seal it, and then dried at room temperature. It is then sealed and stored away from light.

[0080] DPTPA-NPs@Anti-Human IgG were sprayed onto a glass cellulose membrane (as above, preferably at a spraying amount of 4 µL / cm). 2 After drying at room temperature, store in a sealed container away from light.

[0081] NC membrane, glass cellulose membrane (i.e.) Figure 1 The DPTPA-NPs@Anti-Human IgG labeling pad, sample pad, and absorbent pad are attached to the backing plate in sequence, with each layer overlapping by 1-2 mm. The assembled backing plate with each component is cut using a strip cutter and finally placed into the plastic casing of the test strip to complete the assembly.

[0082] (4) Dynamokinetic analysis of DPTPA-NPs@Anti-Human IgG immunoreactivity:

[0083] The test strips were tested, with the T-line and C-line fluorescence values ​​measured every minute. The T / C ratio was calculated by recording the fluorescence intensity of the T-line and C-line over 30 minutes to plot an immunodynamic curve. The T / C ratio was plotted on the ordinate, and time on the abscissa, providing suggested testing times. Figure 11 As shown, the T / C value tends to level off after about 14 minutes of reaction, and a reaction time of 15 minutes is recommended for the test strip.

[0084] (5) Establishment of the standard curve for the AIE-enhanced PreS1 antibody fluorescent test strip:

[0085] HB2 antibody standards were diluted in multiple gradients with healthy human serum for testing, with three parallel experiments for each concentration. A standard curve was plotted with the concentration of positive samples with added HB2 antibody as the x-axis and the T / C value as the y-axis, giving the quantitative range of HB2 antibody detection for this test strip. HB2 antibody standards were diluted in 17 gradients with healthy human serum for testing, with three parallel experiments for each concentration. The results are as follows: Figure 12 As shown, as the HB2 antibody concentration increased from 15.3 pg / mL to 1000 ng / mL, the T / C ratio also increased. However, at higher concentrations, the ratio decreased, possibly due to the "hook effect." When the detection limit is exceeded, it is recommended to dilute the sample before testing. The standard curve was fitted using a four-parameter logistic model, with the regression equation y = 0.07 + 0.224x + 0.286x. 2 +0.096x 3 The limit of detection (LOD) was calculated to be 0.51 ng / mL based on the average concentration of 17 blank samples plus three times the standard deviation.

[0086] Example 4: Performance testing and analysis of the AIE-enhanced PreS1 antibody fluorescent test strip:

[0087] (1) Evaluation of correctness and repeatability:

[0088] Recovery experiments were conducted to determine the accuracy of the test strips. Three samples with different concentrations were tested, and the recovery rates were calculated. The results are shown in Table 1. The intra-batch recovery rate was between 94.00% and 99.73%, with a coefficient of variation (CV) between 2.8% and 6.4%. The inter-batch recovery rate was between 92.00% and 100.6%, with a CV between 5.5% and 7.3%. The test strips developed in this invention have reliable accuracy and excellent precision.

[0089] Table 1. Accuracy and Precision:

[0090]

[0091] (2) Specificity evaluation:

[0092] HCV serum and mouse antibody were used for testing. After 15 minutes, the color development of the test line (T line) and control line (C line) was observed. The results after HCV and mouse antibody testing are as follows: Figure 13 As shown, no positive signal was displayed on the fluorescent test strip, and the T / C ratio was less than 0.03, indicating that the test strip has good specificity.

[0093] (3) Repeatability evaluation:

[0094] Six batches of test strips were used to test for two different concentrations of HBV genotypes B and C, a mixed standard solution of B and C, and healthy human serum under identical conditions. The test results of the six batches of test strips for each concentration of standard solution were compared, and the repeatability of the test strips was assessed based on the degree of variation in the results. The repeatability test results are shown below. Figure 14 As shown in (a), the results of the six batches of test strips were consistent when interpreted with the naked eye, as... Figure 14 As shown in (b), the detection performance is good, no false positives were found in the serum of healthy people, and the results were consistent with those of samples containing HBV PreS1 antibody.

[0095] (4) Thermal stability evaluation:

[0096] The same batch of prepared test strips were placed in a 37 ℃ incubator, and serum tests were performed before, 3 days after, 7 days after, and 10 days after incubation. The results of the thermal stability test are as follows: Figure 15 As shown, the fluorescent test strips prepared by this invention, when placed at 37 °C for 10 days, showed almost identical color development effects regardless of whether the serum had a high or low PreS1 antibody value. The T / C value fluctuated slightly around 2, indicating good stability.

[0097] (5) Transportation stability evaluation:

[0098] Test strips prepared in the same batch were subjected to transport stability testing. They were shaken on a rocker arm at 60 rpm for 7 days, and then dropped three times from a height of 1.5 meters. Serum samples were tested before and after the simulated transport test. If the T-line was clear and consistent, it was considered to have good transport stability. The transport stability test results are as follows: Figure 16 As shown, the T-line color remained clear and unchanged before and after the transportation simulation experiment, and the readings of the card reader showed no change, indicating that transportation did not affect the performance of the test strip.

[0099] Example 5: Serum test analysis of the AIE-enhanced PreS1 antibody fluorescent test strip:

[0100] Sixty cases were tested using the test strip of this invention, and the results were compared with those of an ELISA quantitative detection kit (ELISA method). The results are as follows: Figure 17 As shown, the correlation coefficient R 2 The value was 0.9838, which is highly consistent with the results of the ELISA quantitative detection kit.

[0101] It should be noted that the AIE-enhanced PreS1 antibody fluorescent test strip and the HBV PreS1 antibody quantitative detection test strip based on AIE molecules mentioned above have the same meaning in the embodiments of the present invention.

[0102] The fluorescent test strip of this invention has a fast detection speed and excellent detection capability. In some remote areas or densely populated cities with limited medical resources, the fluorescent test strip can serve as a powerful detection solution.

[0103] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0104] Those skilled in the art will understand that the purpose of this invention is to provide a relatively universal overall preparation process or technical principle, and to cover as many different applicable scenarios and conditions as possible (such as different raw material characteristics, production scale, product demand, etc.). Therefore, some specific operations can be flexibly adjusted according to the situation in actual implementation, as long as the expected or the same or similar technical effects as those in the embodiments of this invention can be achieved.

[0105] The steps in the method of this invention can be adjusted, combined, or deleted according to actual needs. The technical features can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the embodiments are described. However, as long as the combinations of these technical features do not contradict each other, they should all be considered within the scope of this invention.

[0106] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a quantitative HBV PreS1 antibody test strip based on AIE molecules, characterized in that, include: S1, Synthesize the AIE fluorescent molecule DPTPA-CHO; Add 4-diphenylaminobenzaldehyde to chloroform, then add N-bromosuccinimide in portions, and stir overnight at room temperature; Then, pure water was added and mixed well. Dichloromethane was added to separate the organic phase. The organic phase was washed with saturated sodium chloride solution, dehydrated with sodium sulfate, filtered, and dried by rotary evaporation. The intermediate product 4-[bis(4-bromophenyl)amino]benzaldehyde was obtained by purification with n-hexane as the eluent. 4-[bis(4-bromophenyl)amino]benzaldehyde and 4-pyridylboronic acid were reacted in a mixed solvent of water, ethanol and toluene under the catalysis of tetra(triphenylphosphine)palladium and the protection of an inert gas. Cs2CO3 was added to the system. After the reaction was completed, 4-(bis(4-(pyridin-4-yl)phenyl)amino)benzaldehyde was obtained, denoted as DPTPA-CHO. S2, AIE fluorescent molecule DPTPA-CHO conjugated with Anti-Human IgG antibody; The DPTPA-CHO and DSPE-PEG-NHS obtained in S1 were dissolved in a mixed solution of tetrahydrofuran and water, sonicated and stirred overnight, and then filtered through a filter membrane to obtain DPTPA-NPs. Add DPTPA-NPs and Anti-Human IgG to PBS buffer, react at room temperature, then quench with Tris buffer, and dialyze to remove free DPTPA-NPs to obtain the AIE fluorescent molecule DPTPA-CHO and Anti-Human IgG antibody conjugate product DPTPA-NPs@Anti-Human IgG. S3, Prepare the PreS1 antibody fluorescent test strip with AIE-enhanced luminescence; The detection lines on the NC membrane were sprayed with HBV PreS1 antigen solution, and the control lines were sprayed with HB2 antibody. The sprayed NC membrane was then dried in an oven, then immersed in BSA solution for sealing, and finally dried at room temperature and stored in a sealed, light-protected container. DPTPA-NPs@Anti-Human IgG were sprayed onto a glass cellulose membrane, then dried at room temperature and stored in a sealed container away from light. The NC membrane, glass cellulose membrane, sample pad, and absorbent pad are attached to the backing plate in sequence. The assembled backing plate with each component attached is then cut using a strip cutter. Finally, the strip is placed into the plastic housing of the test strip to complete the assembly.

2. The method for preparing the HBV PreS1 antibody quantitative detection test strip based on AIE molecules according to claim 1, characterized in that, In S1, the molar ratio of 4-diphenylaminobenzaldehyde to N-bromosuccinimide is 1:2.1; the volume ratio of the eluent n-hexane to dichloromethane is 4:5; and the molar ratio of 4-[bis(4-bromophenyl)amino]benzaldehyde to 4-pyridylboronic acid is 33:

80.

3. The method for preparing the HBV PreS1 antibody quantitative detection test strip based on AIE molecules according to claim 1, characterized in that, In S1, the inert gas is argon, and the volume ratio of water:ethanol:toluene in the mixed solvent is 1:1:

2. The reaction conditions are 100 °C with stirring overnight.

4. The method for preparing the HBV PreS1 antibody quantitative detection test strip based on AIE molecules according to claim 1, characterized in that, In S2, the mass ratio of DPTPA-CHO to DSPE-PEG-NHS is 1:5; the molar ratio of DPTPA-NPs to Anti-Human IgG is 5:

1.

5. The method for preparing the HBV PreS1 antibody quantitative detection test strip based on AIE molecules according to claim 1, characterized in that, In S2, during the reaction of DPTPA-NPs with Anti-Human IgG, sodium bicarbonate was used to adjust the pH of the solution to 8.

6. The method for preparing the HBV PreS1 antibody quantitative detection test strip based on AIE molecules according to claim 1, characterized in that, In S2, the ultrasonic treatment time is not less than 1 min; the filter membrane pore size is 0.22 µm; and the reaction time of DPTPA-NPs and Anti-Human IgG at room temperature is not less than 2 h.

7. The method for preparing the HBV PreS1 antibody quantitative detection test strip based on AIE molecules according to claim 1, characterized in that, In S3, the concentration of HBV PreS1 antigen solution used was 1.5–2 mg / mL; the spraying volume of DPTPA-NPs@Anti-HumanIgG was 3–4 µL / cm. 2 .

8. The method for preparing the HBV PreS1 antibody quantitative detection test strip based on AIE molecules according to claim 1, characterized in that, In S3, the NC membrane is sealed by immersing it in a 2% w / v BSA solution.

9. A quantitative test strip for HBV PreS1 antibody based on AIE molecules, characterized in that, The test strip is prepared using the method described in any one of claims 1 to 8 for the quantitative detection of HBV PreS1 antibody based on AIE molecules.

10. The application of the HBV PreS1 antibody quantitative detection test strip based on AIE molecules as described in claim 9 in the preparation of a hepatitis B virus PreS1 antibody detection kit.

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