A method for preparing a fluorescent detection probe, its product and application

Abstract

This invention discloses a method for preparing a fluorescent detection probe, its product, and its application. The preparation method involves synthesizing an aqueous stable CsPbBr3 perovskite, functionalizing it, and then conjugating it with rabbit anti-human IgG. The resulting fluorescent detection probe is used to detect human IgG. Specifically, the CsPbBr3 perovskite is encapsulated using MOFs, and then treated with dopamine hydrochloride to form a polydopamine (PDA) layer on the surface of the aqueous stable CsPbBr3 perovskite, thus functionalizing the material surface. This invention utilizes antigen-antibody specific recognition, using the aqueous stable CsPbBr3 perovskite as a fluorescent detection probe to label antibodies, achieving specific recognition of human IgG. The material preparation method used in this invention is simple, has a wide detection range, high sensitivity, and low detection limit. The detection linearity range of this sensor is 1.0 × 10⁻⁶. ‑4 ~5.0 μg / mL, detection limit is 0.0402 ng / mL.

Description

Technical Field

[0001] This invention relates to a method for preparing a fluorescent detection probe, its product and application, belonging to the field of fluorescent immunoassay technology. Background Technology

[0002] Perovskite quantum dots have attracted widespread attention due to their simple fabrication process, tunable emission wavelength and bandgap, high fluorescence intensity, and fast charge transfer rate. Furthermore, in recent years, this material has shown broad application prospects in optoelectronic devices, optoelectronic detection, and other fields. However, their poor stability, especially their sensitivity to aqueous environments, limits their practical application development under certain conditions.

[0003] In recent years, dopamine has begun to be used as a biomimetic adhesion material. In an alkaline environment, dopamine can undergo oxidative self-polymerization to generate polydopamine (PDA). Studies have shown that the oxidative self-polymerization reaction is effective at pH 8.5, and the resulting PDA film can cover the surfaces of various materials. Because PDA contains N-groups and phenolic hydroxyl groups, materials coated with PDA will have many active groups on their surface, enabling further reactions with the functional groups of compounds or biomolecules without the addition of activators. Currently, enzyme-linked immunosorbent assay (ELISA) is commonly used to detect IgG in serum. Although the absorbance of the enzymatic reaction products can be used for qualitative and quantitative detection of the target analyte, ELISA requires the use of enzyme proteins as markers, and removing interference from other substances in the sample requires a cumbersome pretreatment process. Therefore, a highly sensitive, accurate, and stable method for detecting human IgG is needed. Summary of the Invention

[0004] Objectives of the Invention: The first objective of this invention is to provide a method for preparing a fluorescent detection probe; the second objective is to provide a fluorescent detection probe obtained by this method; and the third objective is to provide an application of this fluorescent detection probe in the detection of human IgG.

[0005] Technical solution: This invention provides a method for preparing a fluorescent detection probe, comprising the following steps:

[0006] (1) Preparation of aqueous-phase stable CsPbBr3 perovskite;

[0007] (2) Functionalize aqueous-phase stabilized CsPbBr3 perovskite;

[0008] (3) Utilizing functionalized aqueous stabilized CsPbBr3 perovskite with rabbit anti-human IgG (Ab2) IgG The fluorescent detection probe is obtained by coupling with the probe.

[0009] In step (1), the preparation of aqueous stable CsPbBr3 perovskite includes the following steps: zinc nitrate hexahydrate, 2-methylimidazole, lead bromide and cesium bromide are mixed and added sequentially to a mixed solution of methanol and N,N-dimethylformamide solution. The mixture is stirred to obtain a mixed solution, centrifuged, washed and vacuum dried to obtain aqueous stable CsPbBr3 perovskite CsPbBr3@ZIF-8.

[0010] The molar ratio of zinc nitrate hexahydrate, 2-methylimidazole, lead bromide, and cesium bromide is (1.0–1.2):(8.0–8.1):1:1.

[0011] The volume ratio of methanol to N,N-dimethylformamide is 9:(1.0 to 1.3).

[0012] The molar ratio of zinc nitrate hexahydrate to methanol was 1:(9~9.2)mmol / mL.

[0013] The stirring reaction time is 5.5 to 8 hours.

[0014] The centrifugation speed was 8000–10000 r, and the centrifugation time was 6–10 min.

[0015] The washing process involves using deionized water.

[0016] Vacuum drying involves drying at 60–65°C for 3–4 hours.

[0017] In step (2), the functionalization of aqueous stable CsPbBr3 perovskite includes the following steps: the aqueous stable CsPbBr3 perovskite is mixed with dopamine hydrochloride in PBS solution at room temperature, centrifuged and washed, and vacuum dried to obtain functionalized aqueous stable CsPbBr3 perovskite.

[0018] The mass ratio of aqueous stable CsPbBr3 perovskite to dopamine hydrochloride is (10.0–10.4):1.

[0019] The concentration of the PBS solution was 0.10–0.12 mol / L.

[0020] The solid-liquid ratio of the aqueous-phase stable CsPbBr3 perovskite to the PBS solution was 10:(1.0~1.2)mg / mL.

[0021] The mixing time is 50 to 60 minutes.

[0022] Vacuum drying involves drying at 60-65℃ for 3-3.5 hours.

[0023] In step (3), the preparation of the fluorescent detection probe includes the following steps:

[0024] (1) Functionalized aqueous stabilized CsPbBr3 perovskite was added to glutaraldehyde PBS solution, stirred and reacted, centrifuged, washed with PBS solution, and vacuum dried to obtain powder.

[0025] (2) Add the powder to rabbit anti-human IgG (Ab2) IgG Incubate in PBS solution, centrifuge, wash with PBS solution, block with BSA solution, wash with PBS solution and prepare a solution to obtain the fluorescent detection probe solution.

[0026] In step (1), the volume concentration of glutaraldehyde in the PBS solution is 5%.

[0027] In step (1), the liquid-to-solid ratio of the functionalized aqueous stable CsPbBr3 perovskite to the glutaraldehyde PBS solution is 100:(5-8) mg / mL.

[0028] In step (1), the stirring reaction time is 1.5 to 2 hours.

[0029] In step (2), the concentration of the PBS solution containing rabbit anti-human IgG is 10–11 μg / mL.

[0030] In step (2), the mass ratio of powder to rabbit anti-human IgG is (500-510):1.

[0031] In step (2), the incubation is carried out at 25°C for 1.5 to 2 hours.

[0032] In step (2), the sealing time with BSA solution is more than 30 minutes.

[0033] The fluorescent detection probe obtained by the preparation method described in this invention.

[0034] The application of the fluorescent detection probe described in this invention in the detection of human IgG.

[0035] The application described in this invention includes the following steps:

[0036] (1) Establishment of logarithmic relationship curves: The enzyme plates were functionalized with dopamine hydrochloride in PBS solution and glutaraldehyde in PBS solution, washed with PBS solution, and then goat anti-rabbit (Ab1) was added. IgGThe PBS solution of BSA was added, incubated, washed with PBS solution, blocked with PBS solution of BSA, washed with PBS solution, PBS solution of different concentrations of antigen human IgG was added, incubated, washed with PBS solution, and then the fluorescent detection probe solution of the present invention was added, incubated at room temperature, and the fluorescence intensity was detected by an ELISA reader. A logarithmic relationship curve was established based on the fluorescence intensity and the concentration of antigen human IgG in PBS solution.

[0037] (2) Sample detection: Following the process in step (1), the actual serum sample diluted with PBS solution was used to replace the PBS solution containing different concentrations of antigen human IgG. The concentration of IgG in the actual serum sample was quantified according to the logarithmic relationship curve.

[0038] In step (1), the concentration of the PBS solution of dopamine hydrochloride is (10.0-10.5) mg / mL.

[0039] In step (1), the amount of PBS solution containing dopamine hydrochloride in each well of the enzyme plate is 200-300 μL.

[0040] In step (1), the functionalization time with dopamine hydrochloride in PBS solution is 3.5 to 4 hours.

[0041] In step (1), the volume concentration of glutaraldehyde in the PBS solution is 5%.

[0042] In step (1), the amount of glutaraldehyde PBS solution used in each well of the enzyme plate is 200-300 μL.

[0043] In step (1), the functionalization treatment with glutaraldehyde in PBS solution takes 1 to 2 hours.

[0044] In step (1), goat anti-rabbit (Ab1) IgG The concentration of the PBS solution was 10–11 μg / mL.

[0045] In step (1), each well of the enzyme plate contains goat anti-rabbit (Ab1) IgG The volume of PBS solution used is 100–110 μL.

[0046] In step (1), goat anti-rabbit (Ab1) is added. IgG The incubation time with PBS solution is 80-90 min.

[0047] In step (1), the concentration of BSA in the PBS solution is (1.0–1.2)%.

[0048] In step (1), the blocking time with BSA in PBS solution is more than 30 minutes.

[0049] The concentrations of different concentrations of human IgG antigen in PBS solutions were 100 μL.

[0050] 1.0×10 -4 μg / mL, 1.0×10 -3 μg / mL, 5.0×10 -3 μg / mL, 1.0×10 -2 μg / mL

[0051] 5.0×10 -2 μg / mL, 1.0×10 -1 μg / mL, 5.0×10 -1 μg / mL, 1.0 μg / mL and 5.0 μg / mL.

[0052] In step (1), the incubation time for adding PBS solutions of different concentrations of antigen human IgG is 90-120 min.

[0053] In step (1), the concentration of the fluorescent detection probe solution is (5-5.2) mg / mL, and the amount of fluorescent detection probe solution used is 100-110 μL per well.

[0054] In step (1), the incubation time at room temperature is 90 to 120 minutes.

[0055] All of the aforementioned PBS solutions had a concentration of 0.10–0.12 mol / L and a pH of 7.4, and were prepared by the user using sodium hydrogen phosphate and disodium hydrogen phosphate.

[0056] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0057] (1) The method for preparing perovskite materials in this invention is simple, easy to operate, and can be used for large-scale preparation;

[0058] (2) The perovskite material used in this invention has a strong fluorescence signal, and the synergistic effect of the metal-organic framework ZIF-8 and 2-methylimidazole gives it good stability in the aqueous phase, which improves the sensitivity and accuracy of detection.

[0059] (3) In this invention, perovskite coated with dopamine hydrochloride is used to prepare a fluorescent detection probe, which has a high success rate in antibody coupling.

[0060] (4) The fluorescent detection probe prepared in this invention has the advantages of wide linear range, high sensitivity, low detection limit, specific recognition, and high throughput detection when applied to the detection of human IgG content. Attached Figure Description

[0061] Figure 1 Scanning electron microscope images of CsPbBr3@ZIF-8 and CsPbBr3@ZIF-8@PDA;

[0062] Figure 2 Transmission electron microscopy images of CsPbBr3@ZIF-8 and CsPbBr3@ZIF-8@PDA;

[0063] Figure 3 The fluorescence spectrum of CsPbBr3@ZIF-8;

[0064] Figure 4 The image shows the FT-IR characterization of the material in Example 1.

[0065] Figure 5 The fluorescence spectra of CsPbBr3@ZIF-8@PDA in detecting different concentrations of human IgG;

[0066] Figure 6 Linearity graph of CsPbBr3@ZIF-8@PDA in detecting different concentrations of human IgG;

[0067] Figure 7 This is a diagram showing the effect of the fluorescent detection probe on the specific recognition of human IgG.

[0068] Figure 8 The effect of preparing fluorescent probes under different conditions to detect human IgG. Detailed Implementation

[0069] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0070] The PBS solution has a concentration of 0.1 mol / L and a pH of 7.4.

[0071] Example 1

[0072] 1. Preparation of aqueous-phase stable CsPbBr3 perovskite: 1 mmol zinc nitrate hexahydrate, 8 mmol 2-methylimidazole, 1 mmol lead bromide, and 1 mmol cesium bromide were successively added to a mixture of 9 mL methanol and 1 mL N,N-dimethylformamide solution to dissolve the solids. The mixture was vigorously stirred with a magnetic stirrer at room temperature for 6 h to obtain a milky white solution. This milky white solution was centrifuged at 8000 rpm for 6 min. The product was washed three times with deionized water and finally vacuum dried at 60 °C for 3 h to obtain aqueous-phase stable CsPbBr3 perovskite.

[0073] (CsPbBr3@ZIF-8).

[0074] 2. Functionalization of aqueous-phase stable CsPbBr3 perovskite: Weigh 100 mg of CsPbBr3@ZIF-8 and 10 mg of dopamine hydrochloride using an electronic balance. Pour both solids into a glass sample vial and add 10 mL of PBS solution. Stir gently with a magnetic stirrer at room temperature for 60 min to obtain a grayish-white solution. Centrifuge the grayish-white solution, wash with PBS solution, and vacuum dry at 60°C for 3 h to obtain the functionalized aqueous-phase stable CsPbBr3 perovskite (CsPbBr3@ZIF-8@PDA).

[0075] 3. Preparation of fluorescent detection probe: Weigh 100 mg of the functionalized aqueous stable CsPbBr3@ZIF-8@PDA solid using an electronic balance, and add 5.5 mL of 5% glutaraldehyde in PBS solution (pH = 7.4, prepared using PBS solution) as a cross-linking agent. Stir gently with a magnetic stirrer at room temperature for 2 hours to obtain a grayish-white solution. Centrifuge the grayish-white solution, wash with PBS solution, and finally vacuum dry at 60°C for 3 hours to obtain the powder treated with glutaraldehyde cross-linking agent. Weigh 5 mg of the glutaraldehyde cross-linking agent powder and add it to 1 mL of 10 μg / mL rabbit anti-human IgG (Ab2). IgG Mix the solutions (prepared with PBS) and incubate the mixture in a water bath at 25°C for 2 hours. Centrifuge and wash twice with PBS. Then, to block unreacted activating groups, add 300 μL of 1% BSA solution (pH = 7.4, prepared with 0.1 mol / L PBS) to the centrifuge tube and seal for 30 minutes, then wash twice with PBS. Prepare 1 mL of the fluorescent detection probe (CsPbBr3@ZIF-8@PDA@Ab2) with PBS. IgG The solution was then stored in the test tube at 4°C for later use.

[0076] Scanning electron microscopy analysis was performed on the CsPbBr3@ZIF-8 and CsPbBr3@ZIF-8@PDA obtained in this embodiment, and the results are as follows: Figure 1 As shown. Figure 1 The images show scanning electron microscope (SEM) images of CsPbBr3@ZIF-8 and CsPbBr3@ZIF-8@PDA, where A is the SEM image of CsPbBr3@ZIF-8 and B is the SEM image of CsPbBr3@ZIF-8@PDA. Figure 1 As can be seen from A, the strong adsorption properties of the synthesized CsPbBr3@ZIF-8 likely caused most of the materials to adhere together, exhibiting a cross-layered structure. This is due to... Figure 1 Comparison of A and B, by Figure 1As can be seen from B, there are many coatings on the surface of the material, which can be considered as dopamine hydrochloride forming polydopamine coating on the surface of the material.

[0077] Transmission electron microscopy analysis was performed on the CsPbBr3@ZIF-8 and CsPbBr3@ZIF-8@PDA obtained in this embodiment, and the results are as follows: Figure 2 As shown. Figure 2 Transmission electron microscopy (TEM) images of CsPbBr3@ZIF-8 and CsPbBr3@ZIF-8@PDA are shown, where A is the TEM image of CsPbBr3@ZIF-8 and B is the TEM image of CsPbBr3@ZIF-8@PDA. Figure 2 As shown in Figure A, the morphology of CsPbBr3@ZIF-8 is a regular blocky rod shape, with a size of approximately 500 nm. No CsPbBr3 QDs were observed in the figure, suggesting that the ZIF-8 MOFs completely encapsulate them, thus preventing their observation in the image. Figure 2 Comparison of A and B, by Figure 2 As shown in Figure B, the CsPbBr3@ZIF-8@PDA surface has a coating, indicating that the surface of the CsPbBr3@ZIF-8 material has been coated with polydopamine, thus achieving surface functionalization of CsPbBr3. Furthermore, perhaps due to surface functionalization, the shape of CsPbBr3@ZIF-8 tends towards a slightly circular shape.

[0078] The fluorescence spectrum of CsPbBr3@ZIF-8 obtained in this embodiment is as follows: Figure 3 As shown. Figure 3 It can be seen that CsPbBr3@ZIF-8 has stability and fluorescence performance in PBS. It can exist stably in PBS, and the fluorescence intensity signal is stable and strong. When irradiated with ultraviolet light with an excitation wavelength of 365nm, CsPbBr3@ZIF-8 shows a fluorescence peak at 518nm and emits green light.

[0079] The CsPbBr3@ZIF-8, CsPbBr3@ZIF-8@PDA, and CsPbBr3@ZIF-8@PDA@Ab2 obtained in this embodiment IgG Infrared spectroscopy analysis was performed, and the results were as follows: Figure 4 As shown. Figure 4 The image shows the FT-IR characterization of the material in Example 1. Comparing the FT-IR characterizations of CsPbBr3@ZIF-8 and CsPbBr3@ZIF-8@PDA, the results are as follows: Figure 4 It can be seen that at 2984cm -1 -3421cm -1The broad absorption band is caused by the stretching vibrations of -OH and -NH, which is attributed to the functionalization of CsPbBr3@ZIF-8 by dopamine hydrochloride. This confirms the presence of PDA and the formation of CsPbBr3@ZIF-8@PDA. Meanwhile, from... Figure 4 It can be seen from CsPbBr3@ZIF-8@PDA@Ab2 IgG At 1600cm -1 -1700cm -1 The FI-TR band is the amide I band, which is not found in CsPbBr3@ZIF-8 and CsPbBr3@ZIF-8@PDA materials, confirming the presence of rabbit anti-human IgG (Ab2). IgG The coupling between CsPbBr3@ZIF-8@PDA successfully prepared a fluorescent detection probe.

[0080] Example 2: Fluorescent detection probes used to detect human IgG levels.

[0081] (1) Add 200 μL of 10 mg / mL dopamine hydrochloride in PBS solution to each well of a 96-well polystyrene plate and let it stand for 4 hours. Then pour out the solution, wash twice with PBS solution, and pat dry.

[0082] (2) Add 200 μL of 5% glutaraldehyde PBS solution to each well of the 96-well polystyrene plate treated with dopamine hydrochloride, and let it stand at room temperature for 2 hours. Pour out the solution from the plate, wash twice with PBS solution, and pat dry.

[0083] (3) Add 100 μL of 10 μg / mL goat anti-rabbit (Ab1) to each well of the 96-well polystyrene plate treated in step (2). IgG The solution was incubated in PBS at room temperature for 90 minutes, and then washed four times with 300 μL of PBS solution.

[0084] (4) Add 300 μL of 0.1 mol / L PBS solution containing 1% BSA (pH = 7.4) to each well of the 96-well polystyrene plate treated in step (3), block at room temperature for 30 minutes, and then wash twice with 300 μL PBS solution.

[0085] (5) Add different concentrations (100 μL 1.0 × 10⁻⁶) of different concentrations to each well of the 96-well polystyrene plate treated in step (4). -4 μg / mL, 1.0×10 -3 μg / mL, 5.0×10 -3 μg / mL, 1.0×10 -2 μg / mL, 5.0×10 -2 μg / mL, 1.0×10-1 μg / mL, 5.0×10 -1 PBS solutions of human IgG antigen (1.0 μg / mL, 1.0 μg / mL, and 5.0 μg / mL) were incubated at room temperature for 120 minutes, followed by washing four times with 300 μL PBS solution. Three replicates were prepared for each concentration.

[0086] (6) Add 100 μL of CsPbBr3@ZIF-8@PDA@Ab2 prepared in Example 1 to each well of the 96-well polystyrene plate treated in step (5). IgG The solution was incubated at room temperature for 90 minutes, and then washed four times with 300 μL PBS solution.

[0087] (7) The signal intensity changes of the fluorescent detection probes in PBS solutions containing different concentrations of human IgG antigen were measured using an ELISA reader at an excitation wavelength of 365 nm. A graph showing the relationship between different concentrations and fluorescence signal intensity was obtained. A curve was plotted with the logarithm of concentration (lgC) on the x-axis and fluorescence signal intensity (F) on the y-axis, and the curve was linearly fitted. The results are as follows: Figure 5-6 As shown.

[0088] Figure 5 The fluorescence spectra of CsPbBr3@ZIF-8@PDA in detecting different concentrations of human IgG; by Figure 5 It can be seen that the higher the concentration of human IgG after washing, the higher the concentration of CsPbBr3@ZIF-8@PDA-labeled Ab2. IgG The more IgG is captured, the stronger the fluorescence intensity signal. Within a certain range, the fluorescence intensity gradually increases with the increase of human IgG concentration. Figure 6 The graph shows the linear relationship between CsPbBr3@ZIF-8@PDA and different concentrations of human IgG. Figure 6 It can be seen that in the linear range of 1.0 × 10 -4 ~5.0 μg / mL. Based on the fitting results, the regression equation is y = 148.90788x + 862.98007, and the linear correlation coefficient is R. 2 =0.9962, detection limit is 0.0402 ng / mL (S / N=3). The error bar is derived from the standard deviation of three measurements.

[0089] Example 3

[0090] To evaluate the specificity of the fluorescent probe, common interfering substances in serum samples, such as CEA, ALP, and Glocose, were selected. The fluorescent probe was incubated with 100 ng / mL CEA and Glocose standard solutions, as well as 100 U / L ALP standard solutions. The results are as follows: Figure 7 As shown. Figure 7This is a graph showing the effect of the fluorescent detection probe on the specific recognition of human IgG. The error bars are derived from the standard deviation of three measurements. Figure 7 As shown, compared to the fluorescence intensity of 100 ng / mL human IgG, the fluorescence intensity of all other interfering substances was relatively small and negligible. This demonstrates that the fluorescent detection probe can specifically bind to human IgG and cause a change in fluorescence intensity signal. Other interfering substances could not bind due to the lack of specific binding sites. This indicates that the fluorescent detection probe has satisfactory specificity for human IgG recognition.

[0091] To evaluate the feasibility of using the prepared aqueous stable CsPbBr3 fluorescent detection probe in human serum samples (from Subei People's Hospital, Yangzhou City), IgG levels in three serum samples were determined using a standard recovery method. The accuracy and sensitivity of the method were evaluated and verified by the recovery rate and relative standard deviation. The results are shown in Table 1.

[0092] Table 1. Detection of IgG concentration in different serum samples by the fluorescent detection probe constructed in this invention.

[0093] Table 1 shows that the recovery rate of human IgG in serum samples ranged from 94.03% to 107.52%, with an RSD of less than 7.42%. These results indicate that the prepared fluorescent detection probe has high performance for practical sample analysis.

[0094] Its accuracy allows it to be applied to the quantitative detection of IgG in actual serum samples.

[0095] Example 4

[0096] This embodiment is basically the same as Example 1, except that in step 1, 1.1 mmol zinc nitrate hexahydrate, 8.1 mmol 2-methylimidazole, 1 mmol lead bromide, and 1 mmol cesium bromide were added sequentially to a mixture of 9 mL methanol and 1.2 mL N,N-dimethylformamide solution to dissolve the solid. The solution was vigorously stirred with a magnetic stirrer at room temperature for 6.5 h to obtain a milky white solution. This milky white solution was centrifuged at 9000 rpm for 6 min. The product was washed three times with deionized water and finally vacuum dried at 63°C for 3.5 h to obtain an aqueous stable CsPbBr3 perovskite (CsPbBr3@ZIF-8).

[0097] Example 5

[0098] This embodiment is basically the same as Embodiment 1, except that the amounts of CsPbBr3@ZIF-8@PDA and rabbit anti-human IgG (Ab2) in step 3 are adjusted respectively. IgGThe binding time of rabbit anti-human IgG (Ab2) was 30 min, 60 min, 90 min, 120 min, 180 min, and 240 min, and the binding time of rabbit anti-human IgG (Ab2) was adjusted. IgG The concentrations of ) were 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL and 25 μg / mL. Figure 8 The image shows the effect of preparing fluorescent probes under different conditions for detecting human IgG. Among them, Figure 8 A represents CsPbBr3@ZIF-8@PDA and rabbit anti-human IgG (Ab2). IgG Combined with the time optimization graph, Figure 8 B represents rabbit anti-human IgG (Ab2). IgG Optimization plot of concentration. (From...) Figure 8 As can be seen from A, with the interaction of CsPbBr3@ZIF-8@PDA and rabbit anti-human IgG (Ab2) IgG The binding time of ) increases, resulting in the formation of CsPbBr3@ZIF-8@PDA@Ab2 IgG The greater the fluorescence intensity, the better. When the reaction time is 90 min, the generated CsPbBr3@ZIF-8@PDA@Ab2 IgG The fluorescence intensity reached its maximum value. Prolonging the binding time did not significantly alter the fluorescence intensity, which may be due to the interaction between CsPbBr3@ZIF-8@PDA and rabbit anti-human IgG (Ab2). IgG The combination of ) reaches a saturation state. (By...) Figure 8 B shows that, with the rabbit anti-human IgG (Ab2) IgG As the concentration of ) increases, the generated CsPbBr3@ZIF-8@PDA@Ab2 IgG The greater the fluorescence intensity, the better. When the concentration is 10 μg / mL to 20 μg / mL, CsPbBr3@ZIF-8@PDA@Ab2 IgG The fluorescence intensity is relatively stable. Therefore, CsPbBr3@ZIF-8@PDA and rabbit anti-human IgG (Ab2) IgG The binding time of ) was 90 min, and it was similar to that of rabbit anti-human IgG (Ab2). IgG The concentration of the bound element was 10 μg / mL.

[0099] Comparative Example

[0100] The performance of the fluorescent detection probe prepared in Example 2 for detecting human IgG was compared with that of the comparative human IgG sensor. The performance testing method was the same as in Example 2, and the specific results are shown in Table 2. The comparative examples 1-5 are respectively cited from the following references.

[0101] Comparative Example 1: Qu Q, Wang J, Zeng C, Wang M, Qi W, He Z. AuNP array coated substrate for sensitive and homogeneous SERS-immunoassay detection of human immunoglobulin G. RSC Advances. 2021;11(37):22744-50.

[0102] Comparative Example 2: Song P, Liu Q, Zhang Y, Liu W, Meng M, Yin Y, et al. The chemical redox modulated switch-on fluorescence of carbon dots for probing alkaline phosphatase and its application in an immunoassay. Rsc Advances. 2018;8(1):162-9.

[0103] Comparative Example 3: Wu L, Li X, Shao K, Ye S, Liu C, Zhang C, et al. Enhanced immunoassay for porcine circovirus type 2 antibody using enzyme-loaded and quantum dots-embedded shell-core silica nanospheres based on enzyme-linked immunosorbent assay. Analytica Chimica Acta. 2015;887:192-200.

[0104] Comparative Example 4: Zhu L, Cui X, Wu J, Wang Z, Wang P, Hou Y, et al. Fluorescence immunoassay based on carbon dots as labels for the detection of human immunoglobulin G. Analytical Methods. 2014;6(12):4430-6.

[0105] Comparative Example 5: Wang Q, Wang BT. Surface plasmon resonance biosensor based on graphene oxide / silver coated polymer cladding silica fiber. Sensors and Actuators B-Chemical. 2018; 275:332-8.

[0106] Table 2. Performance comparison of the fluorescence detection method constructed in this invention with the human IgG detection methods in Comparative Examples 1-5

[0107]

[0108]

[0109] As shown in Table 2, the fluorescent detection probe prepared by this invention is significantly superior to the methods for detecting human IgG in Comparative Examples 1-5.

[0110] This invention proposes a simple and environmentally friendly method for preparing aqueous stable CsPbBr3. Unlike other methods, this method synthesizes aqueous stable CsPbBr3 in a one-pot process, eliminating the need for precursor solution synthesis. Furthermore, this method does not require solvents such as toluene, making it more environmentally friendly. Perovskite materials with good stability and high fluorescence intensity can be obtained directly through centrifugal water treatment. The synthesis method of this invention is simple to operate and has good reproducibility. Compared with other methods, in the preparation of fluorescent detection probes, the fluorescent material needs to have carboxyl groups, and subsequent activation with coupling agents results in a low coupling success rate. In this invention, dopamine hydrochloride is used to functionalize the material, forming polydopamine on the material surface, and a crosslinking agent is added to improve the coupling success rate.

Claims

1. A method for preparing a fluorescent detection probe, characterized in that, Includes the following steps: (1) Preparation of aqueous stable CsPbBr3 perovskite includes the following steps: zinc nitrate hexahydrate, 2-methylimidazole, lead bromide and cesium bromide are mixed and added sequentially to a mixed solution of methanol and N,N-dimethylformamide. The mixture is stirred to obtain a mixed solution, centrifuged, washed and vacuum dried to obtain aqueous stable CsPbBr3 perovskite CsPbBr3@ZIF-8; wherein, the molar ratio of zinc nitrate hexahydrate, 2-methylimidazole, lead bromide and cesium bromide is 1.0~1.2:8.0~8.1:1:1, the volume ratio of methanol and N,N-dimethylformamide is 9:1.0~1.3, and the molar ratio of zinc nitrate hexahydrate to methanol is 1 mmol:9~9.2 mL; (2) Functionalizing aqueous-phase stable CsPbBr3 perovskite includes the following steps: mixing aqueous-phase stable CsPbBr3 perovskite with dopamine hydrochloride in PBS solution at room temperature, centrifuging and washing, and vacuum drying to obtain functionalized aqueous-phase stable CsPbBr3 perovskite; wherein, the mass ratio of aqueous-phase stable CsPbBr3 perovskite to dopamine hydrochloride is 10.0~10.4:1, the concentration of PBS solution is 0.10~0.12mol / L, and the solid-liquid ratio of aqueous-phase stable CsPbBr3 perovskite to PBS solution is 10 mg:1.0~1.2 mL; (3) A fluorescent detection probe was obtained by coupling functionalized aqueous stable CsPbBr3 perovskite with rabbit anti-human IgG, including the following steps: (A1) Functionalized aqueous-phase stable CsPbBr3 perovskite was added to a glutaraldehyde PBS solution, stirred and reacted, centrifuged, washed with PBS solution, and vacuum dried to obtain powder; wherein, the volume concentration of glutaraldehyde PBS solution was 5%, and the solid-liquid ratio of functionalized aqueous-phase stable CsPbBr3 perovskite to glutaraldehyde PBS solution was 100 mg: 5~8 mL; (A2) Add the powder to the PBS solution of rabbit anti-human IgG, incubate, centrifuge, wash with PBS solution, block with BSA solution, wash with PBS solution and prepare a solution to obtain the fluorescent detection probe; wherein, the concentration of the PBS solution of rabbit anti-human IgG is 10~11μg / mL, and the mass ratio of powder to rabbit anti-human IgG is 500~510:

1.

2. The method for preparing the fluorescent detection probe according to claim 1, characterized in that, In step (1), the stirring reaction time is 5.5~8h, the centrifugation time is 6~10min, the washing is done with deionized water, and the vacuum drying is done at 60~65℃ for 3~4h.

3. The method for preparing the fluorescent detection probe according to claim 1, characterized in that, In step (2), the stirring time is 50-60 min, and the vacuum drying is carried out at 60-65℃ for 3-3.5 h.

4. The method for preparing the fluorescent detection probe according to claim 1, characterized in that, In step (A1), the stirring reaction time is 1.5~2h; in step (A2), the incubation is carried out at 25℃ for 1.5~2h, and the blocking time with BSA solution is more than 30min.

5. The fluorescent detection probe obtained by the preparation method according to any one of claims 1-4.

6. The application of the fluorescent detection probe of claim 5 in the detection of human IgG, wherein the application is not for disease diagnosis purposes.

7. The application according to claim 6, characterized in that, Includes the following steps: (1) Establishment of logarithmic relationship curve: The enzyme plate was functionalized with PBS solution of dopamine hydrochloride and PBS solution of glutaraldehyde, washed with PBS solution, patted dry, added with PBS solution of goat anti-rabbit, incubated, washed with PBS solution, blocked with PBS solution of BSA, washed with PBS solution, added with PBS solution of different concentrations of antigen human IgG, incubated, washed with PBS solution, then added fluorescent detection probe, incubated at room temperature, and the fluorescence intensity was detected by microplate reader. Logarithmic relationship curve was established based on fluorescence intensity and concentration of antigen human IgG. (2) Sample detection: Following the process in step (1), the actual serum sample diluted with PBS solution was used to replace the PBS solution containing different concentrations of human IgG antigen. The concentration of IgG in the actual serum sample was quantified based on the logarithmic relationship curve.

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