A method for detecting GPC3 based on fluorescence resonance energy transfer to construct a fluorescence biosensor

Abstract

The application discloses a method for detecting GPC3 based on a fluorescence resonance energy transfer type fluorescence biosensor, and uses GPC3 aptamer as a recognition probe, wherein the GPC3 aptamer can specifically recognize and combine with GPC3 protein; and the method is based on the fluorescence resonance energy transfer principle among boron, nitrogen doped carbon quantum dots BNCQDs-GPC3 aptamer and two-dimensional metal organic framework Cu-TCPP(Fe) to establish a fluorescence biosensor for detecting GPC3, so as to detect the content of GPC3 in human serum. The method simplifies the whole detection process according to one-step reaction principle, has a short detection cycle time and low cost.

Description

Technical Field

[0001] This invention belongs to the field of optical sensing technology, specifically relating to a method for detecting GPC3 using a fluorescent biosensor based on fluorescence resonance energy transfer. Background Technology

[0002] Golgi protein 73 (GPC3) is a tumor marker. Patent CN 114113604A discloses a method for detecting the GPC3 marker based on flow cytometry. This method uses magnetic fluorescently encoded microspheres as carriers to conjugate the fluorescent label with a target antibody, forming a fluorescent immunoassay complex. The target concentration is calculated by detecting the fluorescence intensity of the complex. However, the preparation of the magnetic fluorescently encoded microspheres used in this method is cumbersome and inconvenient. Patent CN 111273033A discloses a GP73 assay kit that combines magnetic microparticle technology with acrid ester labeling technology to achieve chemiluminescent determination of the target. This technology requires a low temperature environment and a long incubation time when conjugating the GPC3 antibody with the target. In fluorescence detection, it is necessary to construct novel fluorescent biosensors with high detection accuracy based on the fluorescence resonance energy transfer (FRET) phenomenon. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method for detecting GPC3 using a fluorescence resonance energy transfer (FRET) biofluorescent sensor based on boron and nitrogen-doped carbon quantum dots (BNCQDs) and copper-node tetra(4-carboxyphenyl)iron porphyrin metal-organic framework (Cu-TCPP(Fe)), with a detection range of 2.5 ng / mL to 200 ng / mL.

[0004] To address this technical challenge, BNCQDs were used as fluorescent materials, and BNCQDs were combined with an amino-modified GPC3 aptamer (GPC3... Apt They bind via amide bonds to form fluorescently labeled BNCQDs-GPC3. Apt Complex. In BNCQDs-GPC3 Apt Add Cu-TCPP(Fe) and BNCQDs-GPC3 Apt The complex binds to Cu-TCPP(Fe) via van der Waals forces and π-π conjugation, undergoing fluorescence resonance energy transfer (FRET). This reduces the overall fluorescence intensity of the system, resulting in the formation of BNCQDs-GPC3. Apt / Cu-TCPP(Fe)FRET fluorescent biosensor; after the addition of GPC3 protein, due to GPC3 Apt Regarding the specificity of GPC3, GPC3 preferentially interacts with BNCQDs-GPC3. Apt Combined to form BNCQDs-GPC3Apt The -GPC3 complex, separated from Cu-TCPP(Fe), suppresses fluorescence resonance energy transfer, thereby enabling BNCQDs-GPC3 Apt The fluorescence was recovered. Based on the changes in the degree of fluorescence intensity recovery in the system, a linear relationship between the fluorescence intensity recovery rate and the GPC3 concentration was established, realizing rapid, sensitive, and selective quantitative detection of GPC3.

[0005] To solve this technical problem, the present invention is carried out according to the following steps: Step 1: Fluorescence resonance donor BNCQDs-GPC3 Apt Preparation (1) Preparation of BNCQDs: Weigh citric acid (C6H8O7) and boric acid (H3BO3) and add pure water to make up to volume and stir evenly; then add ethylenediamine monohydrate (C2H8N2O·H2O), heat, cool and dialyze; filter the dialyzed solution, and freeze-dry the filtered solution to obtain nitrogen-doped carbon quantum dots (BNCQDs) solid; (2) BNCQDs-GPC3 Apt Preparation: Nitrogen-doped carbon quantum dots (BNCQDs) and GPC3 aptamers were measured. Apt The crosslinking was activated using EDC / NHS crosslinking agent, and the mixture was stirred and incubated at room temperature in the dark to obtain BNCQDs-GPC3. Apt Solution.

[0006] Step 2: Construction of a fluorescence biosensor based on fluorescence resonance energy transfer (1) Weigh tetra(4-carboxyphenyl)porphyrin iron(III) (TCPP(Fe)) powder and dissolve it completely in a mixed solution of N,N-dimethylformamide (DMF) and ethanol in a volume ratio of 3:1 to obtain a tetra(4-carboxyphenyl)porphyrin iron(III) (TCPP(Fe)) dispersion; (2) Weigh copper nitrate trihydrate (Cu(NO3)) 2• Dissolve 3H2O), trifluoroacetic acid (C2HF3O2), and polyvinylpyrrolidone (PVP) powder thoroughly in a mixed solution of N,N-dimethylformamide (DMF) and ethanol; (3) The tetra(4-carboxyphenyl)porphyrin iron(III) (TCPP(Fe)) dispersion and the obtained mixed solution were mixed, sonicated, heated and reacted to obtain a copper node tetra(4-carboxyphenyl)ironporphyrin metal-organic framework (Cu-TCPP(Fe)) solution; the obtained Cu-TCPP(Fe) solution was centrifuged, washed and dried to obtain Cu-TCPP(Fe); 1 mg of Cu-TCPP(Fe) was weighed and added to 1 mL of pure water and stirred to obtain a Cu-TCPP(Fe) dispersion with a concentration of 1 mg / mL; (4) Mix 100 μL Cu-TCPP(Fe) solution with 200 μL BNCQDs-GPC3 Apt The solutions are mixed; incubated; the fluorescence of BNCQDs is quenched to form BNCQDs-GPC3. Apt / Cu-TCPP(Fe)FRET fluorescent biosensor; scanned with a fluorescence spectrophotometer, with a fixed excitation wavelength of 365 nm, the fluorescence intensity at 455 nm was measured and denoted as F0; Step 3: Plotting the GPC3 working curve (1) Add GPC3 solutions of different concentrations to the obtained fluorescent biosensor, incubate, and detect the peak change at 455 nm; scan with a fluorescence spectrophotometer, fix the excitation wavelength at 365 nm, measure the fluorescence intensity at 455 nm, and record it as F1; (2) Plot the working curve using (F1-F0) / F0 as the ordinate and GPC3 concentration as the abscissa, and calculate the lowest detection limit of the fluorescent biosensor. Step 4: Detection of GPC3 in actual samples (1) Add the actual sample to be tested to the fluorescent biosensor obtained in step 2, incubate, scan with a fluorescence spectrophotometer, fix the excitation wavelength at 365 nm, and record the fluorescence intensity at 455 nm. (2) Calculate the concentration of GPC3 in the actual sample to be tested based on the working curve of GPC3 obtained in step 3.

[0007] As a preferred option: In step 1, the amount of citric acid is 2.5 g and the amount of boric acid is 1.0 g; In step 1, after adding pure water, the volume is adjusted to 15 mL; In step 1, 1 mL of ethylenediamine monohydrate is added; In step 1, the heating temperature is 200℃ and the reaction time is 8 h; In step 1, the centrifugation is performed at a speed of 10,000 rpm for 10 minutes. In step 1, dialysis is performed using a dialysis bag with a molecular weight of 500 at 4°C for 48 hours. In step 1, filtration is performed using a 0.22 μm microporous membrane. The concentration of BNCQDs in step 1 is 1.0 mg / mL; GPC3 in step 1 Apt The DNA sequence is 5'-NH2-C6-GCAGTTGATCCTTTGGATACCCTGG-3'; In step 1, the crosslinking agent solution contains carbodiimide (EDC) and N-hydroxysuccinimide (NHS), wherein the concentration of EDC is 0.7668 mg / mL and the concentration of NHS is 2.1713 mg / mL. In step 1, the BNCQDs solution and GPC3 Apt The volume ratio of the solution to the crosslinking agent solution is 10:10:3; In step 1, the incubation temperature is 35°C and the time is 70 min. The mass of TCPP(Fe) in step 2 is 5.0 mg; In step 2, 4 mL of a mixed solution of N,N-dimethylformamide (DMF) and ethanol in a volume ratio of 3:1 is added; In step 2, 10 mg of copper nitrate trihydrate (Cu(NO3)) is weighed out. 2• 3H2O), 10 μL trifluoroacetic acid (C2HF3O2) and 10 mg polyvinylpyrrolidone (PVP); In step 2, 4 mL of a mixed solution of N,N-dimethylformamide (DMF) and ethanol in a volume ratio of 3:1 is added. In step 2, the volume ratio of TCPP(Fe) solution to the mixed solution is 1:1; In step 2, the ultrasonic time after mixing the TCPP(Fe) solution and the mixed solution is 10 min. The reaction in step 2 is heated at 80°C for 15 hours. In step 2, centrifugation is performed at a speed of 10000 r / min for 10 min; In step 2, the concentration of Cu-TCPP(Fe) solution is 1.0 mg / mL; In step 2, the Cu-TCPP(Fe) solution and BNCQDs-GPC3 Apt The volume ratio of the liquids is 1:1; In step 2, the incubation temperature is 25°C and the time is 50 min; In steps 2, 3 and 4, the excitation wavelength of the fluorescence spectrophotometer is 365 nm and the emission wavelength is 455 nm. In steps 3 and 4, the incubation temperature is 35°C and the time is 50 min.

[0008] Step 1 provides a nitrogen-doped carbon quantum dot (BNCQDs) that emits blue fluorescence and a BNCQDs-GPC3. Apt The probe provides a fluorescent donor for the fluorescence resonance energy transfer reaction system in step 2. Step 2 provides Cu-TCPP(Fe) nanomaterials as the fluorescence energy transfer acceptor; due to BNCQDs-GPC3 Apt There are van der Waals forces and π-π conjugation between BNCQDs and Cu-TCPP(Fe), making BNCQDs-GPC3 Apt Approaching Cu-TCPP(Fe) triggers the FRET process, leading to fluorescence quenching of the system. Step 3 is a further extension of step 2; when GPC3 protein is added to BNCQDs-GPC3... Apt When using the / Cu-TCPP(Fe)FRET fluorescence sensor, due to GPC3 Apt The original conformation of BNCQDs-GPC3 is altered by preferentially combining with GPC3. Apt The interaction force between BNCQDs and Cu-TCPP(Fe) is greatly weakened. Therefore, BNCQDs-GPC3 Apt Separation from Cu-TCPP(Fe) interrupts the FRET process, thereby allowing BNCQDs-GPC3 to separate. Apt The fluorescence recovery was observed. The working curve of GPC3 in step 3 provides the basis for the calculation of GPC3 concentration in the actual sample in step 4. It is evident that steps 1-4 support each other and work together to utilize BNCQDs-GPC3. Apt A fluorescence resonance energy transfer mechanism between GPC3 and Cu-TCPP(Fe) was established, and BNCQDs-GPC3 capable of detecting GPC3 was constructed. Apt / Cu-TCPP(Fe)FRET fluorescent biosensor.

[0009] Compared with the prior art, the present invention has the following advantages: 1. By leveraging the strong fluorescence quenching ability of Cu-TCPP(Fe) and the high stability of BNCQDs, the error of the fluorescent biosensor is reduced, and its accuracy is improved. Cu-TCPP(Fe) itself has a high specific surface area and abundant functional groups, exhibiting strong affinity and effectively adsorbing more aptamer chains, thus enhancing the quenching ability of the fluorescent donor BNCQDs and improving the detection range of the fluorescent biosensor (2.5 ng / mL~200 ng / mL). The novel fluorescent biosensor constructed based on the principle of fluorescence resonance energy transfer (FRET) features a simple detection process, short detection cycle, wide detection range, strong anti-interference capabilities, and strong adaptability. Attached Figure Description

[0010] Figure 1 A schematic diagram illustrating the principle of the BNCQDs / Cu-TCPP(Fe) FRET fluorescent biosensor for detecting GPC3, constructed based on BNCQDs and Cu-TCPP(Fe).

[0011] Figure 2 BNCQDs and BNCQDs-GPC3 Apt The ultraviolet-visible absorption spectrum.

[0012] Figure 3 Image A is a transmission electron microscope (TEM) image of BNCQDs; image B is a scanning electron microscope (SEM) image of Cu-TCPP(Fe).

[0013] Figure 4 BNCQDs-GPC3 at different GPC3 concentrations Apt Fluorescence recovery intensity map of the / Cu-TCPP(Fe)FRET fluorescent biosensor.

[0014] Figure 5 The working curve was plotted with (F1-F0) / F0 as the ordinate and GPC3 concentration as the abscissa. Detailed Implementation

[0015] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0016] A method for detecting GPC3 using a fluorescent biosensor based on BNCQDs / Cu-TCPP(Fe) fluorescence resonance energy transfer binding aptamer is described below. The detection principle is as follows: Figure 1 First, BNCQDs are used as fluorescent groups. Activated BNCQDs are then reacted with GPC3 containing an amino group. Apt They bind via CO-NH bonds to form fluorescently labeled BNCQDs-GPC3. AptProbe; then add Cu-TCPP(Fe). Due to the strong conjugation between Cu-TCPP(Fe) and BNCQDs, Cu-TCPP(Fe) and BNCQDs-GPC3 are combined. Apt Closely approximating, it reveals BNCQDs-GPC3 Apt Fluorescence quenching; after the addition of the target GPC3 protein, GPC3... Apt It specifically binds to the GPC3 protein, thereby enabling BNCQDs-GPC3 Apt Keep away from Cu-TCPP(Fe), BNCQDs-GPC3 Apt The fluorescence was restored, establishing a fluorescent biosensor for detecting GPC3 protein. By measuring changes in fluorescence intensity using a fluorescence spectrophotometer, quantitative analysis of GPC3 protein can be effectively achieved.

[0017] The specific implementation steps are as follows: Step 1: BNCQDs-GPC3 Apt Preparation (1) 2.5 g citric acid and 1.0 g boric acid were diluted with pure water to 15 mL. After stirring until completely dissolved, 1 mL of ethylenediamine monohydrate solution was slowly added dropwise. Then, the solution was placed in a high-temperature reactor lined with polytetrafluoroethylene and heated in a forced-air drying oven at 200 °C for 8 h to obtain unpurified BNCQDs solution.

[0018] (2) Centrifuge the obtained BNCQDs solution at 10,000 rpm for 10 min; place the supernatant into a dialysis bag with a molecular weight cutoff of 500 and dialyze at 4°C for 48 h; filter the dialysis solution through a 0.22 μm microporous membrane; then place the filtered solution in a vacuum drying oven at 50°C for 12 hours to obtain BNCQDs solid; (3) Weigh 10 mg of the obtained BNCQDs solid, add pure water, and make up to 10 mL to obtain a 1 mg / mL BNCQDs solution. Weigh 7.668 mg EDC and 2.1713 mg NHS, mix them, add pure water, and make up to 10 mL. Stir well to obtain a crosslinking agent solution. Measure 50 μL of BNCQDs and 50 μL of 2 μM GPC3. Apt Mix with 30 μL of crosslinking agent solution, place in a 35°C dark environment, shake well, and incubate for 70 min to obtain the fluorescently labeled composite BNCQDs-GPC3. Apt Solution. BNCQDs were characterized using a Shimadzu UV-2600i and a Thermo-Fishe F200XG2. Figure 2 It is BNCQDs and BNCQDs-GPC3Apt UV-Vis absorption spectrum of BNCQDs-GPC3 Apt The absorption peak of BNCQDs red-shifts, indicating that BNCQDs are related to GPC3. Apt Connection successful. Figure 3 A is a transmission electron microscope (TEM) image of BNCQDs, showing that the prepared BNCQDs are uniform in size, well dispersed, and have a particle size of about 7 nm.

[0019] Step 2: Construction of a fluorescent biosensor based on the principle of fluorescence resonance energy transfer (1) Weigh 5 mg of tetra(4-carboxyphenyl)porphyrin ferric chloride using a precision electronic balance, and dissolve it completely in a mixed organic solvent system of 4 mL DMF and ethanol in a volume ratio of 3:1 to obtain TCPP(Fe) dispersion; (2) Weigh 10 mg of copper nitrate trihydrate, 10 μL of trifluoroacetic acid and 10 mg of polyvinylpyrrolidone using a precision electronic balance, and dissolve them completely in a mixed organic solvent system of 4 mL of DMF and ethanol in a volume ratio of 3:1 to obtain a mixed solution. (3) After adding TCPP(Fe) dispersion dropwise to the mixed solution, the mixture was sonicated for 10 min to dissolve. The mixture was then transferred to a reaction vessel and heated at 80 °C for 15 h. After the reaction was completed, the mixture was cooled to room temperature, and the mixed liquid was placed in a centrifuge and centrifuged at 10000 r / min for 10 min. The precipitate was washed three times with anhydrous ethanol and dried under vacuum at low temperature to obtain a dark brown Cu-TCPP(Fe) solid. 1 mg of Cu-TCPP(Fe) solid was weighed and diluted to 1 mL with pure water to obtain a 100 mg / mL Cu-TCPP(Fe) solution. Cu-TCPP(Fe) was characterized using a Hitachi SU8020 scanning electron microscope (SEM). Figure 3 B shows a scanning electron microscope (SEM) image of Cu-TCPP(Fe). The image reveals that Cu-TCPP(Fe) has a two-dimensional lamellar structure with a large overall material specific surface area, which is beneficial for BNCQDs-GPC3. Apt Adsorption; (4) Take 100 μL of 1 mg / mL Cu-TCPP(Fe) and 100 μL of 2 μM BNCQDs-GPC3 Apt After mixing and shaking to ensure homogeneity, incubate at 25°C for 50 min to obtain BNCQDs-GPC3. Apt / Cu-TCPP(Fe) FRET fluorescent biosensor. Scanning was performed using an F-320 fluorescence spectrophotometer with a fixed excitation wavelength of 365 nm. The fluorescence intensity at 455 nm was measured and denoted as F0.

[0020] Step 3: Plotting the GPC3 working curve BNCQDs-GPC3 after the fluorescence intensity was measured in step 2 Apt The / Cu-TCPP(Fe) FRET fluorescent biosensor was evenly divided into 8 groups, and then 200 μL of GPC3 protein solution (2.5 ng / mL, 5.0 ng / mL, 10.0 ng / mL, 20.0 ng / mL, 40.0 ng / mL, 80.0 ng / mL, 100 ng / mL, 200 ng / mL) was added according to the concentration gradient. After shaking and mixing, the mixture was reacted at 35℃ for 50 min. The fluorescence intensity at 455 nm was measured using a fluorescence spectrophotometer with a fixed excitation wavelength of 365 nm, and denoted as F1. BNCQDs-GPC3 at different GPC3 concentrations... Apt The fluorescence spectrum of the / Cu-TCPP(Fe) FRET fluorescent biosensor is shown below. Figure 4 ,Depend on Figure 4 It can be seen that the fluorescence recovery intensity ((F1-F0) / F0) of the fluorescent biosensor is positively correlated with the GPC3 concentration. When the GPC3 protein concentration ranges from 2.5 ng / mL to 200 ng / mL, BNCQDs-GPC3... Apt The fluorescence recovery value of the / Cu-TCPP(Fe)FRET fluorescent biosensor showed a linear relationship with the GPC3 concentration, such as... Figure 5 As shown, the working curve is Y = 0.00284X + 0.5407 (Y represents fluorescence recovery intensity, X represents the concentration of GPC3 protein), and the correlation coefficient is R. 2 =0.9968. The limit of detection was calculated to be 1.239 ng / mL using the formula LOD=3Sd / b (where Sd represents the standard deviation of three blank groups and b represents the slope of the working curve).

[0021] Step 4: Detection of GPC3 in actual serum samples Serum samples were analyzed using a spiked method. Three serum samples were collected: normal human serum, serum from patients with cirrhosis, and serum from patients with hepatocellular carcinoma. The serum samples were diluted 10-fold with phosphate-buffered saline (PBS). GPC3 protein was then added to the diluted serum to final concentrations of 10.0 ng / mL, 40.0 ng / mL, and 100.0 ng / mL, respectively. Each serum sample was measured in triplicate. Serum samples were obtained from the Department of Laboratory Medicine at the 924th Hospital of the Chinese People's Liberation Army (Guilin, China) and were analyzed in accordance with the requirements of the Ethics Committee of the 924th Hospital.

[0022] Take 400 μL of serum sample containing GPC3 protein and add it to the prepared BNCQDs-GPC3. AptThe / Cu-TCPP(Fe)FRET fluorescent biosensor was thoroughly mixed and incubated for 50 min, and then its fluorescence intensity was measured. Each serum sample was measured three times. Based on the working curve Y=0.00284X+0.5407 obtained in step 3, the concentration of GPC3 in the corresponding actual serum sample was calculated. The detection results are shown in Table 1. Table 1 shows that BNCQDs-GPC3... Apt The Cu-TCPP(Fe)FRET fluorescent biosensor showed recoveries of 95.42%–113.30% and relative standard deviations of 0.28%–5.93% for the detection of GPC3 in serum, indicating that the fluorescent biosensor can be applied to the detection of actual serum samples.

[0023] Table 1. Detection results of GPC3 in actual serum samples

[0024] Sample 1: Serum from a healthy person; Sample 2: Serum from a patient with cirrhosis; Sample 3: Serum from a patient with liver cancer.

Claims

1. A method for detecting GPC3 using a fluorescence biosensor constructed based on fluorescence resonance energy transfer for non-diagnostic and / or therapeutic purposes, characterized in that, Follow these steps: Step 1: Fluorescence resonance donor boron and nitrogen-doped carbon quantum dots-GPC3 aptamers BNCQDs-GPC3 Apt Preparation (1) Preparation of boron and nitrogen doped carbon quantum dots (BNCQDs): Weigh citric acid and boric acid, add pure water to make up to volume and stir, then add ethylenediamine monohydrate and stir evenly, heat, cool, centrifuge and dialyze; filter with microporous membrane; vacuum dry the filtered solution to obtain solid BNCQDs; (2) BNCQDs-GPC3 Apt Preparation: Measure BNCQDs and GPC3 aptamer GPC3 Apt The cross-linking agent was used for activation and cross-linking, followed by incubation to obtain BNCQDs-GPC3. Apt Solution; Step 2: Construction of a fluorescence biosensor based on fluorescence resonance energy transfer (1) Weigh TCPP(Fe) powder, add N,N-dimethylformamide DMF and ethanol mixed solution to make up the volume, disperse TCPP(Fe) powder in mixed solvent to obtain TCPP(Fe) dispersion; (2) Measure copper nitrate trihydrate, trifluoroacetic acid and polyvinylpyrrolidone, add N,N-dimethylformamide DMF and ethanol mixed solution to make up to volume, and obtain mixed solution; (3) Mix the TCPP(Fe) dispersion and the mixed solution, sonicate, heat, cool, and centrifuge; wash and dry the separated solids; Cu-TCPP(Fe) solid was obtained; the Cu-TCPP(Fe) solid was weighed, pure water was added and stirred to obtain Cu-TCPP(Fe) dispersion; (4) Mix Cu-TCPP(Fe) solution and BNCQDs-GPC3 Apt The solutions are mixed and incubated to quench the fluorescence of BNCQDs, forming BNCQDs-GPC3. Apt / Cu-TCPP(Fe)FRET fluorescent biosensor; scanned with a fluorescence spectrophotometer, with a fixed excitation wavelength of 365 nm, the fluorescence intensity at 455 nm was measured and denoted as F0; Step 3: Plotting the GPC3 working curve (1) Add GPC3 solutions of different concentrations to BNCQDs-GPC3 Apt / Cu-TCPP(Fe)FRET fluorescent biosensor, incubated, and peak change at 455 nm was detected; scanned with a fluorescence spectrophotometer, with the excitation wavelength fixed at 365 nm, the fluorescence intensity at 455 nm was measured and recorded as F1; (3) Using (F1-F0) / F0 as the ordinate and GPC3 concentration as the abscissa, plot the working curve and calculate the lowest detection limit of the fluorescent biosensor. Step 4: Detection of GPC3 in actual samples (1) Add the actual sample to be tested to the fluorescent biosensor obtained in step 2, incubate, scan with a fluorescence spectrophotometer, fix the excitation wavelength at 365 nm, and record the fluorescence intensity at 455 nm. (2) Calculate the concentration of GPC3 in the actual sample to be tested based on the working curve of GPC3 obtained in step 3.

2. The method according to claim 1, characterized in that: The dialysis described in step 1 is performed using a dialysis bag with a molecular weight cutoff of 500, and the dialysis time is 48 hours.

3. The method according to claim 1, characterized in that: The concentration of BNCQDs in step 1 was 1.0 mg / mL; GPC3 Apt The concentration was 2.0 μM; the crosslinking agent solution contained carbodiimide EDAC and N-hydroxysuccinimide NHS, with EDAC concentration of 0.7668 mg / mL and NHS concentration of 2.1713 mg / mL.

4. The method according to claim 1, characterized in that: In step 1, BNCQDs solution and GPC3 Apt The volume ratio of the solution to the crosslinking agent solution was 10:10:3, and the incubation time was 70 min.

5. The method according to claim 1, characterized in that: In step 2, the volume ratio of DMF to ethanol is 3:1; the heating time is 15 h.

6. The method according to claim 1, characterized in that: The Cu-TCPP(Fe) solution and BNCQDs-GPC3 mentioned in step 2 Apt The volume ratio of the liquid to the incubator was 1:1, the incubation temperature was 35°C, and the incubation time was 70 min.

7. The method according to claim 1, characterized in that: In step 3, the incubation temperature is 25°C and the incubation time is 50 min; in step 4, the incubation temperature is 35°C and the incubation time is 50 min.

8. The method according to claim 1, characterized in that: In step 4, the incubation temperature is 35°C and the incubation time is 50 minutes.

Images