Ratio fluorescence detection method for GP73 based on Zn-TCPP (Fe) nano material

Abstract

The invention relates to a ratio fluorescence detection method of GP73 based on Zn-TCPP (Fe) nanometer, which is characterized in that an NGQDs / Zn-TCPP (Fe)-GP73Apt / DAP ratio fluorescence aptamer sensor is constructed by taking NGQDs as a fluorescence donor, DAP as a fluorescence receptor and Zn-TCPP (Fe)-GP73Apt as an identification probe and a catalyst. The specific binding of GP73 and GP73Apt enables the catalase-like activity of Zn-TCPP (Fe) to be recovered, and more DAPs are generated in the same time. Due to the FRET phenomenon between the NGQDs and the DAP, the DAP fluorescence intensity (F562) is enhanced, the NGQDs fluorescence intensity (F445) is reduced, and the GP73 is quantitatively detected according to the linear relation between the ratio F562 / F445 of the DAP fluorescence intensity and the NGQDs fluorescence intensity and the concentration of the GP73.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to a method for ratio fluorescence detection of GP73 based on nanomaterials combined with aptamers. Background Technology

[0002] Early diagnosis of hepatocellular carcinoma (HCC) is crucial, and Golgi protein 73 (GP73) is an ideal serum biomarker for HCC. GP73 detection methods include enzyme-linked immunosorbent assay (ELISA), Western blotting, magnetic separation ELISA, Luminex multifactor liquid chromatography with fluorescence detection, and electrochemical detection. Among these, fluorescence detection has been widely studied due to its advantages of speed, sensitivity, and convenience. However, the signals of common monochromatic fluorescence sensors are easily affected by light source intensity and environmental factors, leading to large measurement errors and limited sensitivity. Publication CN114965392A discloses a monochromatic fluorescence detection method for GP73 based on NGQDs-MoS2 fluorescence resonance energy transfer combined with aptamer detection, but this method only achieves a detection limit of 1.29 ng / mL. A ratiometric fluorescence sensor needs to be constructed, employing a dual-signal ratio strategy to improve anti-interference and sensitivity. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method for detecting GP73 based on a ratiometric fluorescent aptamer sensor of tetrakis(4-carboxyphenyl)porphyrin zinc iron (Zn-TCPP(Fe)) nanomaterials combined with aptamers, with a detection limit of 0.05 ng / mL.

[0004] To solve this technical problem, Zn-TCPP(Fe)-GP73 was constructed. Apt A sensor combining catalase-like activity with GP73 aptamer-specific recognition function was developed. The sensor, acting as a probe, catalyzes the decomposition of H2O2, oxidizing non-fluorescent o-phenylenediamine (OPD) to orange-yellow fluorescent 2,3-diaminophenazine (DAP). A fluorescence resonance energy transfer (FRET) system was constructed using blue fluorescent nitrogen-doped graphene quantum dots (NGQDs) as the donor and DAP as the acceptor. Upon addition of GP73, aptamer detachment led to the recovery of the activity of Zn-TCPP(Fe) nanomaterials, with decreased fluorescence of NGQDs (445 nm) and increased fluorescence of DAP (562 nm). This FRET was then transmitted via F73. 562 / F 445 The linear relationship between the ratio and GP73 concentration enables highly sensitive detection.

[0005] This invention is carried out according to the following steps:

[0006] Step 1: Preparation of fluorescent molecules NGQDs

[0007] An improved method for preparing NGQDs as disclosed in CN116858902A was developed. Citric acid and urea were dissolved in 5-15 mL of ultrapure water at a molar ratio of 1:3-6 and stirred. The solution was then transferred to a high-pressure reactor and subjected to hydrothermal reaction at 140-200℃ for 2-24 h. After cooling, the solution was centrifuged and dialyzed with a dialysis bag with a molecular cutoff of 300-3000 Da for 8-24 h. The solution was then dried to obtain pure NGQDs powder.

[0008] Step 2: Zn-TCPP(Fe)-GP73 Apt Synthesis

[0009] (1) Preparation of Zn-TCPP(Fe) nanomaterials: Referring to the literature, zinc nitrate hexahydrate (Zn(NO3)2·6H2O), polyvinylpyrrolidone (PVP), and pyrazine were dissolved in 12 mL of dimethylformamide / ethanol (DMF / EtOH) mixture. 4 mL of DMF / EtOH mixture containing tetracarboxyphenylporphyrin iron (TCPP(Fe)) was added to the above mixed solution. After ultrasonic treatment, the resulting mixture was transferred to a high-pressure reactor for hydrothermal reaction. After the reaction, the liquid in the reactor was removed, centrifuged, and the precipitate was collected and washed with anhydrous ethanol. The collected precipitate was dried to obtain brown Zn-TCPP(Fe) nanomaterials.

[0010] (2) Zn-TCPP(Fe)-GP73 Apt Construction: The Zn-TCPP(Fe) nanomaterials prepared in step 2 (1) were ultrasonically crushed to prepare a Zn-TCPP(Fe) nanomaterial solution of 0.3~2.0 mg / mL; then mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC / NHS) crosslinking agent at a molar ratio of 2~4:1 at a volume ratio of 10:1~3, and ultrasonically activated the carboxyl groups on the Zn-TCPP(Fe) nanomaterials; then the activated Zn-TCPP(Fe) nanomaterial solution was mixed with 4~12 μM amino-modified GP73 Apt The solutions were mixed at a volume ratio of 1:1~2 and incubated with shaking at 25~45℃ for 1~12 h. Zn-TCPP(Fe)-GP73 was obtained through an amide reaction between amino and carboxyl groups. Apt Solution, refrigerate.

[0011] Step 3: Plotting the GP73 standard working curve

[0012] (1) Mix standard GP73 solutions of different concentrations with Zn-TCPP(Fe)-GP73 AptMix the solutions at a volume ratio of 1 to 2:1 and incubate at a temperature of 4 to 45°C for 20 to 60 minutes.

[0013] (2) Add acetate buffer (HAc-NaAc) to the mixed solution in step 3(1) to adjust the pH of the solution to 4~6.5. Then add 1.2~2.2 mg / mL NGQDs, 50~500 mM OPD and 50~500 mM H2O2 solution in sequence. Incubate at 4~35℃ in the dark for 10~30 min. Scan with a fluorescence spectrophotometer and record the fluorescence intensity (F) at 445 nm and 562 nm. 445 and F 562 ).

[0014] (3) With F 562 / F 445 Using GP73 concentration as the independent variable and linear fitting as the dependent variable, a standard working curve was obtained, and the limit of detection was calculated.

[0015] Step 4: Detection of GP73 in actual serum samples

[0016] (1) Take an actual serum sample and mix it with the Zn-TCPP(Fe)-GP73 prepared in step 2. Apt Mix the solutions at a volume ratio of 1 to 2:1 and incubate at a temperature of 4 to 45°C for 20 to 60 minutes.

[0017] (2) Add acetate buffer (HAc-NaAc) to the solution in step 4(1) to adjust the pH of the solution to 4~6.5. Then add 1.2~2.2 mg / mL NGQDs, 50~500 mM OPD and 50~500 mM H2O2 solution in sequence. Incubate at 4~35℃ in the dark for 10~30 min. Then scan with a fluorescence spectrophotometer and record the fluorescence intensity (F) at 445 nm and 562 nm. 445 and F 562 ).

[0018] (3) Calculate the concentration of GP73 in the actual serum sample to be tested based on the standard working curve of GP73 obtained in step 3.

[0019] Preferably, in step 1, the volume of ultrapure water is 5 mL;

[0020] Preferably, in step 1, the hydrothermal reaction time is 4 hours.

[0021] Furthermore, in step 1, the centrifugation speed of NGQDs is 12000 r / min, and the centrifugation time is 5 min;

[0022] Preferably, in step 1, the molecular rejection capacity of the dialysis bag is 3000 Da, and the dialysis time is 9 h;

[0023] Furthermore, in step 2, the mass of zinc nitrate hexahydrate is 3.0 mg, the mass of polyvinylpyrrolidone is 20.0 mg, the mass of pyrazine is 0.8 mg, and the volume ratio of DMF / EtOH is 3:1;

[0024] Furthermore, in step 2, the temperature and time of the hydrothermal method are 80℃ and 24 h, respectively, and the speed and time of the centrifuge during centrifugal washing are 10000 r / min and 8 min, respectively.

[0025] Preferably, in step 2, the molar ratio of crosslinking agent EDC / NHS is 2:1;

[0026] Furthermore, in step 2, GP73 Apt The DNA sequence is 5′-NH2-C6-GCAGTTGATCCTTTGGATACCCTGG-3′;

[0027] Preferably, in step 2, Zn-TCPP(Fe)-GP73 is synthesized. Apt GP73 Apt The concentration was 10 μM, the incubation temperature was 35℃, and the incubation time was 12 h. At this time, the peroxidase-like activity of the Zn-TCPP(Fe) nanomaterials was influenced by GP73. Apt The impact is most pronounced;

[0028] Furthermore, in steps 3 and 4, the concentration of the added HAc-NaAc buffer is 0.2 M;

[0029] Preferably, the incubation temperature of GP73 protein in steps 3 and 4 is 35°C and the incubation time is 30 min.

[0030] Furthermore, in steps 3 and 4, the fluorescence excitation wavelength is 365 nm, which allows for the simultaneous and relatively strong excitation of NGQDs and DAPs.

[0031] Preferably, in steps 3 and 4, the pH of the solution is adjusted to 5.5 with HAc-NaAc buffer and the incubation temperature is 25°C in the dark, at which point the efficiency of OPD to DAP conversion is the highest.

[0032] Preferably, in steps 3 and 4, the concentration of NGQDs is 1.8 mg / mL, the concentration of OPD is 100 mM, the concentration ratio of OPD to H2O2 is 1:2, and the incubation time in the dark is 20 min. At this time, the fluorescence intensity of DAP is closest to that of NGQDs, which is beneficial for subsequent detection.

[0033] Step 1 provides an NGQD emitting blue fluorescence, which is the source of the fluorescence signal at 445 nm in step 3. Step 2 provides Zn-TCPP(Fe)-GP73, which has recognition function and peroxidase-like properties. Apt On the one hand, it is the reason why GP73 can be specifically detected in step 3; on the other hand, it accelerates the conversion of OPD to DAP by catalyzing the decomposition of H2O2, and DAP is the source of the fluorescence signal at 562 nm in step 3. When GP73 is absent in the system, NGQDs / Zn-TCPP(Fe)-GP73 Apt The / DAP solution exhibited two emission peaks (445 nm and 562 nm) under 365 nm excitation light. In step 3, with the addition of GP73, GP73 reacted with GP73... Apt Specific binding, GP73 Apt Detachment from the surface of Zn-TCPP(Fe) nanomaterials is a key step in this invention; it leads to enhanced catalase activity in solution, i.e., an increase in DAP concentration; this results in enhanced fluorescence intensity at 562 nm, while NGQDs are further weakened due to DAP quenching of their fluorescence. The higher the GP73 concentration, the more pronounced this change. This establishes the F... 562 / F 445 The linear relationship between Zn-TCPP(Fe)-GP73 concentration and GP73 concentration is established. This linear relationship in step 3 provides the basis for calculating the GP73 concentration in the actual sample in step 4. Steps 1 to 4 support each other and work together to achieve the desired Zn-TCPP(Fe)-GP73 concentration. Apt The aptamer sensor enables rapid, low-cost ratio fluorescence detection of GP73.

[0034] Beneficial effects

[0035] This patent provides a Zn-TCPP(Fe) nanomaterial with a unique morphology and good catalase-like activity, by linking the Zn-TCPP(Fe) nanomaterial to GP73. Apt Generate Zn-TCPP(Fe)-GP73 AptThis sensor simultaneously functions as a recognition probe and catalyst. It specifically recognizes GP73 protein and efficiently catalyzes the decomposition and oxidation of OPD by H2O2, generating DAP with yellow fluorescence as a secondary fluorescent signal. A FRET phenomenon occurs between NGQDs and DAP, causing a decrease in the blue fluorescence of NGQDs and an increase in the yellow fluorescence of DAP. The ratiometric fluorescence sensor constructed in this way effectively counteracts environmental factors such as temperature, pH, light source fluctuations, and background interference, improving detection stability and sensitivity. Within a linear range of 1.0–200.0 ng / mL for GP73 concentration, the linear regression correlation coefficient R² = 0.9913, the limit of detection is 0.05 ng / mL, and the relative standard deviation ranges from 0.26% to 4.30%, thus demonstrating superior performance. Attached Figure Description

[0036] Figure 1 Schematic diagram of a ratiometric fluorescent aptamer sensor for detecting GP73 constructed based on Zn-TCPP(Fe) nanomaterials;

[0037] Figure 2 Characterization of NGQDs: (A) Fluorescence and absorption spectra of NGQDs; (B) High-resolution transmission electron microscopy (HRTEM) characterization of NGQDs.

[0038] Figure 3 Characterization images of Zn-TCPP(Fe) nanomaterials: (A) Scanning electron microscope (SEM) image of Zn-TCPP(Fe) nanomaterials; (B) X-ray photoelectron spectroscopy (XPS) characterization image of Zn-TCPP(Fe) nanomaterials;

[0039] Figure 4 Zn-TCPP(Fe)-GP73 Apt The ultraviolet-visible absorption spectrum;

[0040] Figure 5 Zn-TCPP(Fe) and Zn-TCPP(Fe)-GP73 Apt Analysis of catalase-like activities;

[0041] Figure 6 A ratiometric fluorophore aptamer sensor was used to detect the fluorescence spectra of GP73 at different concentrations. Detailed Implementation

[0042] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0043] A ratiometric fluorescence detection method for GP73 based on Zn-TCPP(Fe) nanomaterials is described below. Figure 1First, NGQDs exhibiting blue fluorescence and Zn-TCPP(Fe) nanomaterials with large specific surface area, abundant active sites, and strong catalase-like activity were prepared separately; then, the Zn-TCPP(Fe) nanomaterials were linked to GP73. Apt As a fluorescent probe and catalyst, it catalyzes the decomposition and oxidation of OPD by H2O2 to generate DAP with yellow fluorescence. In this process, NGQDs act as fluorescent donors and DAP as fluorescent acceptors, exhibiting a FRET phenomenon. Under 365 nm UV excitation, DAP and NGQDs emit the strongest fluorescence at 562 nm and 445 nm, respectively. Zn-TCPP(Fe) nanomaterials are linked to GP73... Apt Generate Zn-TCPP(Fe)-GP73 Apt At this time, due to the reduction in active sites, its catalase-like activity decreases. When GP73 protein is added to the system, GP73... Apt The specific binding of the protein GP73 to the Zn-TCPP(Fe) nanomaterial allows it to detach from the surface, restoring the catalase-like activity of the Zn-TCPP(Fe) nanomaterial. This results in the production of more DAP within the same timeframe, leading to the blue fluorescence (F) of NGQDs in the system. 445 The fluorescence of DAP (F) weakens, and the yellow fluorescence of DAP decreases. 562 The effect is enhanced. The higher the GP73 concentration, the more pronounced this change. Ultimately, based on F... 562 / F 445 The relationship between the ratio of GP73 and its concentration was investigated, and a standard working curve for GP73 was established to achieve rapid and highly sensitive quantitative detection of GP73. The specific implementation steps are as follows:

[0044] Step 1: Preparation of fluorescent molecules NGQDs

[0045] 0.21 g of citric acid and 0.18 g of urea were dissolved in 5 mL of water and stirred to form a clear solution. The clear solution was transferred to a 20 mL Teflon-lined stainless steel autoclave, then transferred to a sealed autoclave and heated to 160°C in a drying oven for 4 hours. After heating, the autoclave was removed and allowed to cool naturally to room temperature. The mixed solution was then centrifuged at 12000 r / min for 5 min to remove water-insoluble impurities. The resulting NGQDs concentrate was dialyzed continuously for 9 hours using a dialysis bag with a molecular cutoff of 3000 Da, with water changed every 3 hours, to remove small molecule impurities and obtain a pure NGQDs stock solution. The pure NGQDs stock solution was dried in a vacuum drying oven at 50°C for 12 hours to obtain black NGQDs powder, which was stored at 4°C for later use. The NGQDs powder was dissolved in pure water and characterized using a fluorescence spectrophotometer and a UV-Vis spectrophotometer. Figure 2(A) shows the fluorescence and absorption spectra of NGQDs, indicating that the excitation and emission wavelengths of NGQDs are 365 nm and 445 nm, respectively. The absorption peaks of NGQDs are 234 nm and 332 nm. The absorption peak at 234 nm is caused by the π-π* transition of C=C, and the absorption peak at 332 nm can be attributed to the n-π* transition of the conjugate CN / C=N part. Figure 2 The HRTEM image in (B) shows that the NGQDs are spherical with a particle size of approximately 3 nm, and the lattice spacing of 0.212 nm corresponds to the (100) crystal plane of the graphite phase. This indicates that the NGQDs have a high degree of graphitization, which gives them a broad spectral response, high fluorescence quantum yield, and tunable luminescence properties. The excellent optical properties of the NGQDs lay the foundation for the detection of GP73.

[0046] Step 2: Zn-TCPP(Fe)-GP73 Apt Synthesis

[0047] (1) Preparation of Zn-TCPP(Fe) nanomaterials:

[0048] 3.0 mg Zn(NO3)2·6H2O, 20.0 mg PVP, and 0.8 mg pyrazine were dissolved in 12 mL of a 3:1 DMF / EtOH mixture. Next, under vigorous stirring, 4 mL of a 3:1 DMF / EtOH mixture containing 4.4 mg TCPP(Fe) was added dropwise to the above mixture. The resulting solution was then sonicated for 10 min, transferred to a Teflon-lined high-pressure reactor, and placed in a forced-air drying oven at 80 °C for 24 h. After the reaction, the reactor was removed and allowed to cool naturally to room temperature. The liquid was then centrifuged at 10000 r / min for 8 min, and the precipitate was collected and washed three times with anhydrous ethanol to remove any unreacted substances. The collected precipitate was then dried in a vacuum drying oven at 50 °C for 12 h to obtain brown Zn-TCPP(Fe) nanomaterials, which were stored at 4 °C for later use. The Zn-TCPP(Fe) nanomaterials were characterized, and the results are as follows: Figure 3 As shown, Figure 3 The SEM image in (A) shows that the synthesized Zn-TCPP(Fe) nanomaterials have obvious wrinkles and a thin film-like structure; Figure 3 The XPS characterization diagram of (B) shows the presence of characteristic peaks of C, O, N, Zn, and Fe, proving that Zn-TCPP(Fe) nanomaterials were successfully synthesized.

[0049] (2) Zn-TCPP(Fe)-GP73 AptConstruction:

[0050] 4 mg of Zn-TCPP(Fe) nanomaterials were dissolved in 8 mL of ultrapure water and sonicated for 1 h to obtain a 0.5 mg / mL Zn-TCPP(Fe) nanomaterial solution. 20 μL of the Zn-TCPP(Fe) nanomaterial solution was taken, and 2 μL of crosslinking agent EDC / NHS (10 mM / 5 mM) was added. The solution was sonicated for 40 min to activate the carboxyl groups. Subsequently, the activated Zn-TCPP(Fe) nanomaterial solution was reacted with 10.0 μM GP73. Apt Equal volumes of solutions were mixed. The mixture was then placed in a constant-temperature shaking incubator at 35°C and 66 rpm for 12 h to obtain Zn-TCPP(Fe)-GP73. Apt Sensor solution. For Zn-TCPP(Fe)-GP73 Apt After centrifugation of the sensor solution, the supernatant was used for UV-Vis absorption spectroscopy characterization, and the results are as follows: Figure 4 As shown, Zn-TCPP(Fe)-GP73 can be seen. Apt Sensor solution supernatant in GP73 Apt The absorbance at the characteristic absorption peak of 260 nm is significantly lower than that of the added GP73. Apt The absorbance indicates that most GP73 Apt Successful attachment to Zn-TCPP(Fe) nanomaterials demonstrates that Zn-TCPP(Fe)-GP73 Apt The sensor was successfully fabricated. Following this, further research was conducted on Zn-TCPP(Fe) nanomaterials and Zn-TCPP(Fe)-GP73. Apt The activities of sensor-based catalases were investigated and compared, and the results are as follows: Figure 5 As shown. From Figure 5 As can be seen, solutions a, b, and c lack the catalyst, H₂O₂, and the colorimetric reagent OPD, respectively, therefore showing no peak at the DAP absorption characteristic peak of 440 nm. Solution d, however, exhibits a significantly higher absorption spectrum than the other three groups, thus verifying the catalase-like activity of Zn-TCPP(Fe). Furthermore, the absorbance of solution e at 440 nm is 0.68, and that of solution d at 440 nm is 1.03, indicating that GP73… Apt The binding with Zn-TCPP(Fe) nanomaterials weakens the catalase-like activity of Zn-TCPP(Fe) nanomaterials, which is the key to this method.

[0051] Step 3: Plotting the GP73 standard working curve

[0052] (1) In 20 μL of the above Zn-TCPP(Fe)-GP73 Apt20 μL of different concentrations of GP73 (1 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng / mL, 150 ng / mL and 200.00 ng / mL) were added to the solution and incubated at 35℃ for 30 min.

[0053] (2) Add 20 μL of 1.8 mg / mL NGQDs, 100 μL of 0.2 M pH 5.5 HAc-NaAc buffer solution, 20 μL of 100.00 mM OPD solution, and 20 μL of 200.00 mM H2O2 solution. Incubate at 25°C in the dark for 20 min. Add 1.8 mL of pure water. Measure the fluorescence intensity (F) at 445 nm and 562 nm using a fluorescence spectrophotometer at an excitation wavelength of 365 nm. 445 and F 562 ).from Figure 6 As can be seen, with the increase of GP73 concentration, the fluorescence intensity at 445 nm decreases, while the fluorescence intensity at 562 nm increases.

[0054] (3) With the concentration of GP73 as the independent variable (X), the ratio of the fluorescence intensity at 445 nm and 562 nm corresponding to each concentration (F) 562 / F 445 With Y as the dependent variable, a linear fitting equation, i.e., the standard working curve, was plotted. Within the linear range of 1.0–200.0 ng / mL, the standard working curve equation for the fluorescence spectrophotometer detection was Y = 0.0054X + 1.4748, with a correlation coefficient of R0. 2 =0.9913.

[0055] (4) Without adding GP73, the blank group (S) was measured using a fluorescence spectrophotometer. blank ) of F 562 / F 445 Therefore, the LOD of GP73 detected by this ratio fluorescence sensor can be calculated to be 0.05 ng / mL.

[0056] Step 4: Detection of GP73 in actual serum samples

[0057] (1) Take 6 actual serum samples, including 2 samples from healthy people, 2 samples from hepatitis patients, and 2 samples from liver cancer patients. Then divide each sample into 3 portions and add 1000 ng / mL standard GP73 solution to each portion, so that the concentration of each sample increases by 0 ng / mL, 5 ng / mL and 10 ng / mL respectively.

[0058] (2) In 20 μL Zn-TCPP(Fe)-GP73 Apt Add 20 μL of the above serum sample to the solution and incubate at 35°C for 30 min.

[0059] (3) Add 20 μL of 1.8 mg / mL NGQDs, 20 μL of 100.00 mM OPD solution, 20 μL of 200.00 mM H2O2 solution, and 100 μL of 0.2 M HAc-NaAc buffer solution with a pH of 5.5. Incubate at 25°C in the dark for 20 min, then add 1.8 mL of pure water. Measure the fluorescence intensity (F) at 445 nm and 562 nm using a fluorescence spectrophotometer at an excitation wavelength of 365 nm. 445 and F 562 ).

[0060] (4) Based on the standard working curve of GP73 obtained in step 3 using the fluorescence spectrophotometer, the concentration of GP73 in the actual serum sample to be tested was calculated. The detection results are shown in Table 1. Table 1 shows that, compared with the clinical method, NGQDs / Zn-TCPP(Fe)-GP73... Apt The relative standard deviation of the / DAP ratio fluorescence sensor for detecting GP73 in serum was between 0.04% and 4.30%, and the recovery rate was between 96.92% and 105.57%, indicating that the constructed ratio fluorescence sensor has good application potential for the determination of GP73 concentration in actual serum samples.

[0061] Table 1. Ratio fluorescence detection results of GP73 in actual serum samples

[0062]

[0063]

Claims

1. A ratiometric fluorescence detection method for GP73 based on Zn-TCPP(Fe) nanomaterials for non-diagnostic / non-therapeutic purposes, characterized in that, Follow these steps: Step 1: Preparation of fluorescent molecules NGQDs Citric acid and urea were dissolved in 5-15 mL of ultrapure water at a molar ratio of 1:3-6 and stirred. The mixture was then transferred to a high-pressure reactor and hydrothermally reacted at 140-200℃ for 2-24 h, followed by cooling. The solution was centrifuged, dialyzed with a dialysis bag with a molecular cutoff of 300-3000 Da for 8-24 h, and then dried to obtain pure NGQDs powder. Step 2: Zn-TCPP(Fe)-GP73 Apt Synthesis (1) Preparation of Zn-TCPP(Fe) nanomaterials: Zinc nitrate hexahydrate Zn(NO3)2·6H2O, polyvinylpyrrolidone PVP and pyrazine were dissolved in 12 mL of dimethylformamide / ethanol DMF / EtOH mixture; 4 mL of DMF / EtOH mixture containing tetracarboxyphenylporphyrin iron TCPP(Fe) was added; the resulting mixture was ultrasonically treated and transferred to a high-pressure reactor for hydrothermal reaction; the liquid in the reactor was removed and centrifuged, the precipitate was collected and washed with anhydrous ethanol; the collected precipitate was dried to obtain brown Zn-TCPP(Fe) nanomaterials; (2) Zn-TCPP(Fe)-GP73 Apt Construction: The Zn-TCPP(Fe) nanomaterials obtained in step 2 (1) were ultrasonically crushed to prepare a solution of 0.3~2.0 mg / mL, and then mixed with a crosslinking agent of 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide EDC / NHS at a molar ratio of 4~2:1 at a volume ratio of 10:1~3, and ultrasonicated; then mixed with 4~12 μM amino-modified GP73 Apt The solutions were mixed at a volume ratio of 1:1~2 and incubated at 25~45℃ for 1~12 h to obtain Zn-TCPP(Fe)-GP73. Apt Solution; Step 3: Plotting the GP73 standard working curve (1) Mix standard GP73 solutions of different concentrations with Zn-TCPP(Fe)-GP73 Apt Mix the solutions at a volume ratio of 1~2:1 and incubate at 4~45℃ for 20~60 min; (2) Add acetate buffer HAc-NaAc to the mixed solution in step 3(1) to adjust the pH of the solution to 4~6.

5. Then add 1.2~2.2 mg / mL NGQDs, 50~500 mM OPD and 50~500 mM H2O2 solution in sequence. Incubate at 4~35℃ for 10~30 min. Scan with a fluorescence spectrophotometer and record the fluorescence intensity F at 445 nm and 562 nm. 445 and F 562 ; (3) With F 562 / F 445 With GP73 concentration as the independent variable and GP73 concentration as the dependent variable, a linear fit was performed to obtain a standard working curve, and the limit of detection was calculated. Step 4: Detection of GP73 in actual serum samples (1) Take an actual serum sample and mix it with the Zn-TCPP(Fe)-GP73 prepared in step 2. Apt Mix the solutions at a volume ratio of 1~2:1 and incubate at 4~45℃ for 20~60 min; (2) Add acetate buffer to the solution in step 4(1) to adjust the pH of the solution to 4~6.5, then add 1.2~2.2 mg / mL NGQDs, 50~500 mM OPD and 50~500 mM H2O2 solution in sequence, incubate at 4~35℃ for 10~30 min, scan with a fluorescence spectrophotometer and record the fluorescence intensity F at 445 nm and 562 nm. 445 and F 562 .

2. The method according to claim 1, characterized in that: In step 1, the molar ratio of citric acid to urea is 1:3, the volume of ultrapure water is 5 mL, the temperature and time of the hydrothermal reaction are 160℃ and 4 h, respectively, the molecular cutoff of the dialysis bag is 3000 Da, and the dialysis time is 6 h.

3. The method according to claim 1, characterized in that: In step 2, the concentration of the Zn-TCPP(Fe) nanomaterial solution was 0.5 mg / mL, the molar ratio of EDC / NHS was 2:1, the volume ratio of Zn-TCPP(Fe) nanomaterial solution to crosslinking agent was 10:1, and the ultrasonic activation time was 40 min.

4. The method according to claim 1, characterized in that: In step 2, GP73 Apt The concentration was 10 μM, and the activated Zn-TCPP(Fe) nanomaterial solution was mixed with GP73. Apt The solution was mixed and incubated at a volume ratio of 1:1, at an incubation temperature of 35℃, for 12 h; GP73 Apt The base sequence is 5′-NH2-C6-GCAGTTGATCCTTTGGATACCCTGG-3′.

5. The method according to claim 1, characterized in that: In step 3, the GP73 solution reacts with Zn-TCPP(Fe)-GP73. Apt The solutions were mixed at a volume ratio of 1:

1.

6. The method according to claim 1, characterized in that: In step 4, the actual serum solution is mixed with Zn-TCPP(Fe)-GP73. Apt The solutions were mixed at a volume ratio of 1:

1.

7. The method according to claim 1, characterized in that: In steps 3 and 4, the pH of the HAc-NaAc buffer was 5.5, the incubation temperature was 25°C, and the incubation time was 20 min.

8. The method according to claim 1, characterized in that: In steps 3 and 4, the concentration of NGQDs was 1.8 mg / mL, the concentration ratio of OPD to H2O2 was 1:2, and the concentration of OPD was set to 100 mM.

9. The dual-color aptamer fluorescent probe according to claim 1, characterized in that: In steps 3 and 4, the excitation wavelength is set to 365 nm, and the emission wavelengths are 445 nm and 562 nm, respectively.

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