A phycoerythrin fluorescent probe, a preparation method and application thereof

Abstract

This invention provides a phycoerythrin fluorescent probe, its preparation method, and its application, belonging to the field of biodetection technology. The invention involves mixing phycoerythrin with the heterobifunctional cross-linking agent SPDP to obtain thiolated phycoerythrin; mixing superoxide dismutase (SOD) with SPDP to obtain pyridine disulfide-modified SOD; and mixing thiolated phycoerythrin with pyridine disulfide-modified SOD and performing a light-protected coupling reaction to obtain the phycoerythrin fluorescent probe. By optimizing the SPDP cross-linking reaction conditions, this invention produces a highly sensitive phycoerythrin-SOD probe with a fluorescence intensity of 3800 MFI and no cytotoxicity. This probe has been successfully applied to an FFA-induced HepG2-NAFLD model, enabling real-time monitoring of SOD activity recovery and lipid metabolism improvement after astaxanthin intervention, providing an efficient tool for the diagnosis and drug screening of oxidative stress-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and in particular relates to a phycoerythrin fluorescent probe, its preparation method and application. Background Technology

[0002] Oxidative stress is a key mechanism in the development of many diseases, with its core being the excessive accumulation of reactive oxygen species (ROS) and the imbalance of the antioxidant system. Traditional detection methods, such as chemiluminescence and ELISA, suffer from low sensitivity and complex operation.

[0003] Phycoerythrin (PE) is a natural fluorescent protein with high quantum yield, large Stokes shift, and good biocompatibility, but its application in the field of fluorescent probes has not been fully explored. Furthermore, there are no reports on phycoerythrin-SOD composite probes and their application in NAFLD models. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a phycoerythrin fluorescent probe, its preparation method and application, to solve the problems of insufficient sensitivity and large background interference of traditional probes, and to realize its application in dynamic monitoring of oxidative stress.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for preparing a phycoerythrin fluorescent probe, comprising the following steps:

[0007] 1) Phycoerythrin and heterobifunctional cross-linking agent SPDP were mixed and reacted in the dark to obtain modified phycoerythrin;

[0008] 2) Mix the DTT solution and the modified phycoerythrin, and react in the dark to obtain thiolated phycoerythrin;

[0009] 3) Superoxide dismutase and heterobifunctional cross-linking agent SPDP were mixed and cross-linked to obtain pyridine disulfide-modified superoxide dismutase.

[0010] 4) Mix thiolated phycoerythrin and pyridine disulfide-modified superoxide dismutase, and perform a light-protected coupling reaction to obtain a phycoerythrin fluorescent probe.

[0011] Preferably, the concentration of phycoerythrin in step 1) is 0.03–0.08 mg / mL; the molar ratio of the heterobifunctional cross-linking agent SPDP to phycoerythrin is 5–640:1;

[0012] The temperature for the light-protected reaction is 22–27°C, and the time for the light-protected reaction is 1–3 hours.

[0013] Preferably, the concentration of the DTT solution in step 2) is 100-200 mmol / L, and the volume ratio of the DTT solution to the modified phycoerythrin is 1:10-50.

[0014] The temperature for the light-protected reaction is 22–27°C, and the time for the light-protected reaction is 20–40 min.

[0015] Preferably, the molar ratio of the heteromorphic bifunctional crosslinking agent SPDP and superoxide dismutase in step 3) is 25 to 800:1.

[0016] Preferably, the temperature of the crosslinking reaction in step 3) is 22-27°C, and the time of the crosslinking reaction is 12-24 hours.

[0017] Preferably, in step 4), the molar ratio of thiolated phycoerythrin to pyridine disulfide-modified superoxide dismutase is 1:1.

[0018] Preferably, the temperature of the light-protected coupling reaction in step 4) is 22-27°C, and the time of the light-protected coupling reaction is 12-24 hours.

[0019] The present invention also provides a phycoerythrin fluorescent probe prepared by the aforementioned preparation method.

[0020] The present invention also provides the phycoerythrin fluorescent probe prepared by the above preparation method or the application of the phycoerythrin fluorescent probe in dynamic monitoring of oxidative stress.

[0021] Preferably, the concentration of the phycoerythrin fluorescent probe is 1–50 μg / mL, and the action time of the phycoerythrin fluorescent probe is 15–120 min.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention optimizes the SPDP crosslinking reaction conditions to prepare a highly sensitive phycoerythrin-SOD probe with a fluorescence intensity of 3800 MFI and no cytotoxicity. This probe was successfully applied to an FFA-induced HepG2-NAFLD model, enabling real-time monitoring of SOD activity recovery and lipid metabolism improvement after astaxanthin intervention, providing an efficient tool for the diagnosis and drug screening of oxidative stress-related diseases.

[0024] The phycoerythrin fluorescent probe of this invention has high sensitivity and can detect changes in SOD activity as low as 10 μg / mL. In the HepG2-NAFLD model induced by 1 mM MFA, the phycoerythrin fluorescent probe successfully detected that SOD activity recovered by 60% after intervention with astaxanthin (20 μM), while TG and TC contents decreased by 56.8% and 63.6%, respectively. Attached Figure Description

[0025] Figure 1 This describes the separation and purification process and spectral characteristics of phycoerythrin;

[0026] Figure 2 The graph shows the spectral effects of different molar ratios of SPDP and R-phycoerythrin on R-phycoerythrin.

[0027] Figure 3 The figure shows the effect of different molar ratios of SPDP and R-phycoerythrin on the fluorescence properties of R-phycoerythrin.

[0028] Figure 4 This is a graph showing the effect of different molar ratios of SPDP and SOD on SOD potency.

[0029] Figure 5 It represents the average fluorescence intensity of cells in each group with different concentrations of phycoerythrin fluorescent probes;

[0030] Figure 6 The average fluorescence intensity of cells in each group at different interaction times with the phycoerythrin fluorescent probe;

[0031] Figure 7 The SOD activity of the phycoerythrin fluorescent probe in the regulation of NAFLD oxidative stress by astaxanthin;

[0032] Figure 8 The levels of TG and TC in the phycoerythrin fluorescent probe are measured in the regulation of NAFLD oxidative stress by astaxanthin. Detailed Implementation

[0033] This invention provides a method for preparing a phycoerythrin fluorescent probe, comprising the following steps:

[0034] 1) Phycoerythrin (PE) and heterobifunctional cross-linking agent SPDP were mixed and reacted in the dark to obtain modified phycoerythrin;

[0035] 2) Mix the DTT solution and the modified phycoerythrin, and react in the dark to obtain thiolated phycoerythrin;

[0036] 3) Superoxide dismutase (SOD) and heterobifunctional cross-linking agent SPDP were mixed and cross-linked to obtain pyridine disulfide-modified superoxide dismutase.

[0037] 4) Mix thiolated phycoerythrin and pyridine disulfide-modified superoxide dismutase, and perform a light-protected coupling reaction to obtain a phycoerythrin fluorescent probe.

[0038] In this invention, phycoerythrin and a heterobifunctional cross-linking agent SPDP are mixed and reacted in the dark to obtain modified phycoerythrin. The concentration of the phycoerythrin is preferably 0.03–0.08 mg / mL, more preferably 0.04–0.07 mg / mL, and even more preferably 0.05 mg / mL; the molar ratio of the heterobifunctional cross-linking agent SPDP to phycoerythrin is preferably 5–640:1, more preferably 10–320:1, and even more preferably 80:1; the temperature of the light-protected reaction is preferably 22–27°C, more preferably 23–26°C, and even more preferably 25°C; the light-protected reaction… The preferred time is 1-3 hours, more preferably 1.5-2.5 hours, and even more preferably 2 hours; after the light-protected reaction, unbound SPDP is removed by ultrafiltration, and the preferred ultrafiltration conditions are: molecular weight cutoff of 5-15 kDa, 4000-4500 rpm, 10-20 min, more preferably: molecular weight cutoff of 8-12 kDa, 4100-4300 rpm, 12-18 min, and even more preferably: molecular weight cutoff of 10 kDa, 4200 rpm, 15 min.

[0039] In this invention, DTT solution and modified phycoerythrin are mixed and reacted in the dark to obtain thiolated phycoerythrin. The concentration of the DTT solution is preferably 100–200 mmol / L, more preferably 120–180 mmol / L, and even more preferably 150 mmol / L; the volume ratio of the DTT solution to the modified phycoerythrin is preferably 1:10–50, more preferably 1:15–45, and even more preferably 1:40; the temperature of the light-protected reaction is preferably 22–27°C, more preferably 23–26°C, and even more preferably 25°C; the time of the light-protected reaction is preferably 20–40 min, more preferably 25–35 min, and even more preferably 30 min; after the light-protected reaction, unbound DTT is removed by ultrafiltration, and the ultrafiltration conditions are preferably: molecular weight cutoff 5–15 kDa, 4000–4500 rpm, 10–20 min, more preferably: molecular weight cutoff 8–12 kDa, 4100–4300 rpm, 12–18 min, and even more preferably: molecular weight cutoff 10 kDa, 4200 rpm, 15 min.

[0040] In this invention, superoxide dismutase and a heterobifunctional cross-linking agent SPDP are mixed and subjected to a cross-linking reaction to obtain pyridine disulfide-modified superoxide dismutase. The molar ratio of the heterobifunctional cross-linking agent SPDP to superoxide dismutase is preferably 25–800:1, more preferably 50–400:1, and even more preferably 80:1; the temperature of the cross-linking reaction is preferably 22–27°C, more preferably 23–26°C, and even more preferably 25°C; the time of the cross-linking reaction is preferably 12–24 h, more preferably 15–21 h, and even more preferably 18 h; after the cross-linking reaction, ultrafiltration purification is performed. The ultrafiltration purification conditions are preferably: molecular weight cutoff 5–15 kDa, 4000–4500 rpm, 10–20 min, more preferably: molecular weight cutoff 8–12 kDa, 4100–4300 rpm, 12–18 min, and even more preferably: molecular weight cutoff 10 kDa, 4200 rpm, 15 min.

[0041] In this invention, thiolated phycoerythrin and pyridine disulfide-modified superoxide dismutase are mixed and coupled in the dark to obtain a phycoerythrin fluorescent probe. The molar ratio of thiolated phycoerythrin to pyridine disulfide-modified superoxide dismutase is 1:1; the temperature of the light-protected coupling reaction is preferably 22-27°C, more preferably 23-26°C, and even more preferably 25°C; the time of the light-protected coupling reaction is preferably 12-24 h, more preferably 15-21 h, and even more preferably 18 h.

[0042] The present invention also provides a phycoerythrin fluorescent probe prepared by the aforementioned preparation method.

[0043] The present invention also provides the phycoerythrin fluorescent probe prepared by the above preparation method or the application of the phycoerythrin fluorescent probe in dynamic monitoring of oxidative stress.

[0044] In this invention, the dynamic monitoring of oxidative stress includes the dynamic monitoring of superoxide dismutase (SOD) activity in a non-alcoholic fatty liver disease (NAFLD) model; the concentration of the phycoerythrin fluorescent probe is preferably 1-50 μg / mL, more preferably 5-20 μg / mL, and even more preferably 10 μg / mL; the action time of the phycoerythrin fluorescent probe is preferably 15-120 min, more preferably 30-60 min.

[0045] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0046] Example 1

[0047] The *Porphyridium cruentum* cultured in a glass column bioreactor was centrifuged three times (each centrifugation at 8000 rpm for 10 min) to obtain a preliminary crude extract. This crude extract was then extracted using ultrasonic disruption (ultrasonic on for 2 seconds, off for 3 seconds, 5 min, power 150 W). Following two-step ammonium sulfate precipitation (25% and 60% saturation) and DEAE-52 ion exchange column chromatography, phycoerythrin (0.052 mg / mL) with a purity ≥4.65 (A565 / A280) was obtained. The separation, purification process, and spectral characteristics of the phycoerythrin are as follows: Figure 1 As shown.

[0048] Desalting was performed using a 0.02 mol / L PBS-EDTA solution.

[0049] An appropriate concentration of SPDP solution (molar ratio of SPDP to PE of 80:1) was added to the PE solution, and the mixture was shaken and reacted for 2 hours at room temperature in the dark to obtain modified phycoerythrin (PE-PDP). Unbound SPDP molecules were removed by ultrafiltration centrifugation (molecular weight cutoff 10 kDa, 4200 rpm, 15 min).

[0050] Mix 25 μL of 150 mmol / L DTT solution with 1 ml of modified phycoerythrin (PE-PDP), and react with shaking at room temperature in the dark for 30 min. Remove excess DTT by ultrafiltration centrifugation (molecular weight cutoff 10 kDa, 4200 rpm, 15 min) to obtain thiolated phycoerythrin (PE-SH).

[0051] SOD and SPDP were reacted at a molar ratio of 1:80 under shaking conditions at room temperature in the dark for 18 h. After ultrafiltration (molecular weight cutoff of 10 kDa, 4200 rpm, 15 min), pyridine disulfide-modified superoxide dismutase (SOD-PDP) was obtained.

[0052] The phycoerythrin fluorescent probe (PE-SOD probe) was prepared by mixing PE-SH and SOD-PDP at a molar ratio of 1:1 and coupling at room temperature in the dark for 18 hours.

[0053] Example 2

[0054] 1. The effect of different molar ratios of SPDP / R-phycoerythrin on the spectrum of R-phycoerythrin.

[0055] R-PE was desalted using 20 mmol / L PBS-EDTA solution. SPDP solutions of different molar concentrations were prepared by dissolving SPDP in dimethyl sulfoxide (DMSO). 500 μL portions of 0.05 mg / mL R-phycoerythrin solution were placed in nine centrifuge tubes. The volume was adjusted to 1 mL with PBS-EDTA. 10 μL of SPDP solution of different molar concentrations was slowly added to each centrifuge tube to establish SPDP to R-phycoerythrin molar ratios of 640, 320, 160, 80, 40, 20, 10, 5, and 0 (control group). After thorough mixing, the mixture was reacted with shaking at room temperature in the dark for 2 h. Unreacted SPDP reagent was removed by ultrafiltration and centrifugation. The solution was then diluted 40-fold with PBS-EDTA, and the absorption spectra of the R-phycoerythrin solutions in each tube were measured.

[0056] Experimental results: such as Figure 2 The effect of different molar ratios of SPDP on the absorption spectrum of R-phycoerythrin is shown. After introducing different amounts of SPDP solution into the R-phycoerythrin solution, significant changes were observed in the UV absorption characteristics of R-phycoerythrin. With increasing molar ratio of SPDP to R-phycoerythrin, the original pink color of R-phycoerythrin gradually faded. The absorbance of R-phycoerythrin at 565 nm gradually decreased, and the absorption peak shifted to a shorter wavelength (blue shift). The absorbance at 490 nm also decreased slightly, but no blue shift was observed. When the molar ratio of SPDP to R-phycoerythrin reached 80, the absorbance at 565 nm decreased by approximately 25% compared to the blank control group. When the molar ratio increased to 640, the absorbance at 565 nm decreased by approximately 40%, and the characteristic absorption peak became less prominent, indicating that the protein may have undergone significant denaturation.

[0057] 2. Effects of different molar ratios of SPDP and R-phycoerythrin on the fluorescence properties of R-phycoerythrin.

[0058] SPDP solutions of varying concentrations were prepared using DMSO. 500 μL of 0.05 mg / mL R-phycoerythrin solution was dispensed into nine centrifuge tubes. The solution volume was adjusted to 1 mL using PBS-EDTA. The predetermined concentrations of SPDP solution were added, ensuring the molar ratios of SPDP to R-phycoerythrin were 640, 320, 160, 80, 40, 20, 10, 5, and 0 (control group). After thorough mixing, the mixture was incubated at room temperature in the dark for 2 hours. Unbound SPDP was removed by ultrafiltration and centrifugation. The relative fluorescence intensity of the reaction solution in each centrifuge tube was measured at the characteristic fluorescence emission wavelength of R-phycoerythrin to evaluate the binding efficiency of SPDP to R-phycoerythrin and its effect on the fluorescence properties of R-phycoerythrin.

[0059] Experimental results: such as Figure 3 The figure shows the effect of different molar ratios of SPDP / R-phycoerythrin on the fluorescence intensity of R-phycoerythrin. The fluorescence properties of R-phycoerythrin are highly sensitive to changes in SPDP concentration. When the molar ratio of SPDP to R-phycoerythrin is below 160, the solution color remains pink. When the molar ratio exceeds 160, the pink color of the solution gradually fades. When the molar ratio reaches 640, the pink color of the solution almost completely disappears, indicating that excessively high SPDP concentrations may lead to overactivation of the reaction, which can adversely affect the structure and properties of the protein.

[0060] 3. The effect of different SPDP / SOD molar ratios on SOD potency

[0061] SOD concentration was set at 0.3 mg / mL. Succinimidyl-2-(2-pyridyldithio)-propionate (SPDP) solutions of different molar concentrations were prepared using DMSO as solvent. Seven 200 μL aliquots were separated from a 2.1 mL 0.3 mg / mL SOD solution. Different concentrations of SPDP solution were added to centrifuge tubes to ensure the SPDP to SOD molar ratio was 0 (control group), 25, 50, 100, 200, 400, and 800. The reaction was carried out at room temperature for 2 h. After the reaction, unbound SPDP molecules were removed by ultrafiltration and centrifugation. SOD activity was assessed using an indirect enzyme-linked immunosorbent assay (ELISA). The enzyme activity of SPDP-modified SOD molecules was detected to evaluate the effect of the SPDP-SOD cross-linking reaction on SOD activity. The regulatory mechanism of SPDP modification on SOD enzyme activity was explored by comparing the changes in SOD activity under different molar ratios.

[0062] Experimental results: such as Figure 4 The effect of SPDP concentration on SOD antibody titer is shown in the figure. Experiments indicate that SPDP significantly inhibits SOD activity, and this inhibitory effect is positively correlated with SPDP concentration. An SPDP / SOD molar ratio of 80 was selected as the optimal concentration ratio. This finding is significant for understanding the role of SPDP in biological or chemical processes. In studies of antioxidant mechanisms or related drug development, the potential impact of SPDP on SOD activity needs to be considered to avoid negative effects.

[0063] Example 3

[0064] Investigation into the optimal conditions for the action of phycoerythrin fluorescent probes.

[0065] HepG2 cells provided by the Shanghai Cell Bank were seeded in 96-well culture plates and cultured for 24 hours to allow them to adhere and grow. The medium was then replaced with serum-free medium containing different concentrations of the phycoerythrin fluorescent probe prepared in Example 1 (0 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL), and incubated under light-protected conditions for 15 min, 30 min, 60 min, and 120 min. After incubation, the medium was discarded and the cells were washed twice with PBS buffer. Medium containing 10% CCK-8 reagent was added and incubated for 2 hours. The optical density (OD) was measured at 450 nm. The average fluorescence intensity of each group of cells was detected and analyzed using flow cytometry.

[0066] Experimental results: such as Figure 5 and Figure 6 As shown. To investigate the optimal probe concentration, an incubation time of 60 min was initially chosen. When the probe concentration was in the range of 1-10 μg / mL, cell viability remained at 93%-98% (vs. control group), with no significant toxicity (p>0.05). When the concentration was ≥20 μg / mL, cell viability significantly decreased to below 85% (p<0.05), indicating that high concentrations of the probe may damage the cell membrane through physical adsorption or oxidative stress. Regarding fluorescence intensity, the fluorescence intensity reached 3800 MFI at 10 μg / mL, and only increased to 4500 MFI at 20 μg / mL (an increase of 18%, with no statistically significant difference). Therefore, 10 μg / mL of probe was selected as the optimal concentration. Further investigation was conducted using 10 μg / mL probe for incubation time. During incubation of 15-60 min, the fluorescence intensity increased from 1200 MFI to 3800 MFI, and cell viability remained >93%. Extending the incubation time to 120 min resulted in only a slight increase in fluorescence intensity (4000 MFI), but cell viability significantly decreased to 85% (p<0.05). The fluorescence intensity plateaued at 60 min, indicating that probe binding to the target site was nearing saturation. Furthermore, prolonged incubation in the dark may inhibit cell metabolism; shortening the incubation time reduces experimental error. Therefore, 60 min was chosen as the optimal incubation time.

[0067] Example 4

[0068] Application of phycoerythrin fluorescent probe in NAFLD model

[0069] 1. Construct a NAFLD model by inducing HepG2 cells with 1mM FFA (PA:OA = 2:1) for 24 hours:

[0070] (1) Accurately weigh 25.6 mg of palmitic acid (PA) and 9.5 mg of oleic acid (OA), dissolve them in 1 mL of anhydrous ethanol, and shake in a water bath at 55 °C until completely dissolved. Mix the FFA ethanol solution with a 10% BSA solution (fatty acid-free) at a volume ratio of 1:9, shake at 37 °C for 1 h to form an FFA-BSA complex. Filter the complex through a 0.22 μm filter membrane for sterilization, and aliquot and store at -20 °C (avoid repeated freeze-thaw cycles).

[0071] (2) NAFLD model induction: 1 mM FFA was used to incubate the cells at 37°C and 5% CO2 for 24 h.

[0072] (3) NAFLD model validation:

[0073] ①Oil Red O absorbance: First, cells were fixed with 4% paraformaldehyde for 15 min and washed 3 times with PBS. Then, they were stained with 0.5% Oil Red O (prepared with isopropanol) for 30 min and washed with PBS to remove excess stain. Finally, the stained lipid droplets were dissolved with isopropanol, and the absorbance was measured at OD510 nm.

[0074] ② Determination of TG and TC content.

[0075] FFA-induced HepG2 cells were collected, washed with PBS, and then lysis buffer (containing 1% Triton X-100) was added. Lysis was performed on ice for 30 min, followed by centrifugation at 12000 rpm for 10 min. The supernatant was used as the test sample. The enzyme working solution (containing lipoprotein lipase, glycerol kinase, etc.) and chromogenic reagent were mixed according to the manufacturer's instructions and used immediately. Blank wells, standard wells, and sample wells were added sequentially to a 96-well plate. The plates were incubated at 37°C in the dark for 10 min, and the absorbance of each well was immediately measured at 510 nm using a microplate reader.

[0076] FFA-induced HepG2 cells were collected, washed with PBS, and then lysed with lysis buffer (containing 1% Triton X-100). Lysis was performed on ice for 30 min, followed by centrifugation at 12000 rpm for 10 min. The supernatant was used as the test sample. Cholesterol esterase, cholesterol oxidase, peroxidase, and chromogenic reagent were prepared into a mixed working solution according to the manufacturer's instructions. Blank wells, standard wells, and sample wells were added sequentially to a 96-well plate. The plate was incubated at 37°C in the dark for 15 min, and the absorbance of each well was immediately measured at 500 nm using a microplate reader.

[0077] 2. Regulation of oxidative stress and lipid metabolism in NAFLD by astaxanthin (Control group: untreated standard HepG2 cells; Model group: NAFLD cell model induced by 1mM FFA; Astaxanthin group: model cells treated with different concentrations of astaxanthin; Positive control group: model cells treated with 50μM silymarin.)

[0078] (1) Add 20 μM astaxanthin for 24 h.

[0079] (2) Add 10 μg / mL of the PE-SOD probe prepared in Example 1 and incubate for 60 min. Detect the MFI value by flow cytometry.

[0080] Experimental results: such as Figure 7 and Figure 8 As shown, SOD activity recovered by 60% (P<0.01), and TG and TC levels decreased to 0.80 mmol / mg (a decrease of 57%) and 0.44 mmol / mg (a decrease of 63.6%), respectively (**p<0.01).

[0081] As demonstrated by the above embodiments, this invention, by optimizing the SPDP crosslinking reaction conditions, produces a highly sensitive phycoerythrin-SOD probe with a fluorescence intensity of 3800 MFI and no cytotoxicity. This probe was successfully applied to the FFA-induced HepG2-NAFLD model, enabling real-time monitoring of SOD activity recovery and lipid metabolism improvement after astaxanthin intervention, providing an efficient tool for the diagnosis and drug screening of oxidative stress-related diseases.

[0082] 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 phycoerythrin fluorescent probe, characterized by, Includes the following steps: 1) Phycoerythrin and heterobifunctional cross-linking agent SPDP were mixed and reacted in the dark to obtain modified phycoerythrin; The molar ratio of the heteromorphic bifunctional crosslinking agent SPDP to phycoerythrin is 5–640:1; 2) Mix the DTT solution and the modified phycoerythrin, and react in the dark to obtain thiolated phycoerythrin; 3) Superoxide dismutase and heterobifunctional cross-linking agent SPDP were mixed and cross-linked to obtain pyridine disulfide-modified superoxide dismutase. The molar ratio of the heterobifunctional cross-linking agent SPDP to superoxide dismutase is 25–800:1; 4) Mix thiolated phycoerythrin and pyridine disulfide-modified superoxide dismutase, and perform a light-protected coupling reaction to obtain a phycoerythrin fluorescent probe.

2. The production method according to claim 1, characterized by, Step 1) The concentration of phycoerythrin is 0.03-0.08 mg / mL; the temperature of the light-protected reaction is 22-27°C; and the time of the light-protected reaction is 1-3 h.

3. The preparation method according to claim 1, characterized in that, Step 2) The concentration of the DTT solution is 100-200 mmol / L, and the volume ratio of the DTT solution to the modified phycoerythrin is 1:10-50; The temperature for the light-protected reaction is 22–27°C, and the time for the light-protected reaction is 20–40 min.

4. The method of claim 1, wherein, Step 3) The cross-linking reaction is carried out at a temperature of 22-27°C for 12-24 hours.

5. The preparation method according to claim 1, characterized in that, In step 4), the molar ratio of thiolated phycoerythrin to pyridine disulfide-modified superoxide dismutase is 1:

1.

6. The method of claim 1, wherein, Step 4) The temperature of the light-protected coupling reaction is 22-27°C, and the time of the light-protected coupling reaction is 12-24 hours.

7. The phycoerythrin fluorescent probe prepared by the preparation method according to any one of claims 1 to 6.

8. The application of the phycoerythrin fluorescent probe prepared by the preparation method according to any one of claims 1 to 6 or the phycoerythrin fluorescent probe according to claim 7 in the preparation of products for dynamic monitoring of oxidative stress.

9. Use according to claim 8, characterized in that, The concentration of the phycoerythrin fluorescent probe is 1–50 μg / mL, and the reaction time of the phycoerythrin fluorescent probe is 15–120 min.

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