A colorimetric-fluorescent dual-mode assay method for hydrophobic anticancer drug curcumin

Abstract

The application provides a colorimetric-fluorescent dual-mode determination method for hydrophobic anticancer drug curcumin, and the application realizes the preparation of oil amine branch joint polysuccinimide loaded curcumin (CUR@PSI OAm ) coated antigen, dual-mode probe labeled anti-PSI OAm antibody, and specific combination of antigen-antibody reaction for dual-mode ultra-trace detection and analysis of CUR@PSI OAm in cells, and indirect quantitative detection of hydrophobic anticancer drug curcumin in cells. Compared with the prior art, the dual-mode analysis method provided by the application can be mutually supported, and does not need to be enriched and preprocessed, is simple to operate, has short detection time, has the characteristics of high throughput, high sensitivity, high specificity, precise targeting and non-destructive detection, and can be used for ultra-trace non-destructive detection and analysis of hydrophobic anticancer drug curcumin (CUR) in cells.

Description

Technical Field

[0001] This invention pertains to drug assay methods, specifically a colorimetric-fluorescence dual-mode assay for the hydrophobic anticancer drug curcumin. Utilizing antigen-antibody specific immunoassay technology, the method indirectly detects intracellular hydrophobic anticancer drug curcumin (CUR) by detecting oleylamine-grafted polysuccinimide nanocarriers loaded with CUR. Background Technology

[0002] Curcumin, a natural hydrophobic polyphenol flavoring extracted from the tropical Southeast Asian plant turmeric, can be used as a food additive, chemical indicator, and in medicine. Numerous studies have shown that curcumin possesses various biochemical functions, including anti-inflammatory, antioxidant, antibacterial, antiviral, anti-proliferative, and anti-mutagenic effects. Furthermore, curcumin exhibits potent anti-tumor activity, inhibiting the proliferation of various tumor cells. Further research indicates that curcumin is a powerful free radical scavenger, making it an ideal radiation protectant for protecting healthy tissues from radiation damage.

[0003] However, the anticancer drug curcumin has poor water solubility and lacks targeting. While it works to kill cancer cells in the human body, it also kills normal cells, leading to serious toxic side effects and limiting the development and application of curcumin in the pharmaceutical field.

[0004] To date, various methods have been developed for the quantitative detection of curcumin, a hydrophobic anticancer drug in cells. The main detection methods include high-performance liquid chromatography (HPLC) – cumbersome pretreatment; liquid chromatography-tandem mass spectrometry (LC-MS / MS) – complex operation steps; Raman spectroscopy – poor repeatability; atomic absorption spectrometry (AAS) – low precision; flow cytometry – expensive instruments; ultraviolet-visible spectrophotometry – low sensitivity; and quantitative nuclear magnetic resonance (qNMR) – long analysis time. These methods have limited the development of highly sensitive detection of curcumin.

[0005] Therefore, it is essential to study a highly sensitive method for the detection of curcumin. Summary of the Invention

[0006] The purpose of this invention is to provide a colorimetric-fluorescence dual-mode assay for the hydrophobic anticancer drug curcumin. This method utilizes antigen-antibody specific immunoassay technology to establish a colorimetric-fluorescence dual-mode detection method. By integrating fluorescence and enzyme catalytic properties onto the same nanoparticle without interference, and using these as detection signals during the experimental process, a dual-mode detection method is simultaneously performed on an oleylamine-grafted polysuccinimide nanoparticle carrier loaded with the hydrophobic anticancer drug curcumin (CUR) within cells. This indirectly detects the intracellular anticancer drug curcumin (CUR) and achieves higher detection accuracy, sensitivity, and specificity.

[0007] The specific technical solution of this invention is as follows:

[0008] A colorimetric-fluorescence dual-mode method for the determination of curcumin, a hydrophobic anticancer drug, includes the following steps:

[0009] a. Preparation of nanomedicine carrier oleylamine-grafted polysuccinimide (PSI) OAm And oleylamine-grafted polysuccinimide-loaded curcumin CUR@PSI OAm Hydrolyzed solution;

[0010] b. Preparation of CUR@PSI OAm Coating antigens and immunogens;

[0011] c. PSI resistance OAm Antibody preparation;

[0012] d. Preparation of dual-mode probes FITC@Pt Janus NPs;

[0013] e. Dual-mode probe FITC@Pt Janus NPs-labeled anti-PSI OAm Antibody preparation;

[0014] f、CUR@PSI OAm Preparation of cell lysate after incubation with HeLa cells;

[0015] g. CUR@PSI OAm The coating antigen was diluted with coating buffer and then coated into 96-well plates. The plates were then blocked, and lysis buffer standards of different concentrations and anti-PSI labeled with a dual-mode probe were added. OAm Antibody, using colorimetric and fluorescent dual-signal molecules to establish a direct competitive dual-mode immunoassay for the intracellular hydrophobic anticancer drug CUR@PSI. OAm ;

[0016] h, with CUR@PSI OAm A standard curve was plotted with the logarithm of the concentration of the lysis buffer standard on the x-axis and the absorbance or fluorescence intensity value on the y-axis. This curve was obtained by grafting polysuccinimide (CUR@PSI) onto an oleylamine nanocarrier loaded with the anticancer drug curcumin (CUR) into the cell. OAm The detection of curcumin (CUR), an intracellular hydrophobic anticancer drug, was indirectly performed.

[0017] Step a specifically includes the following steps:

[0018] a-1, 5-20 mg PSI OAm0.2–5 mg curcumin (CUR) and 0.2–5 mg polyethylene-b-polyethylene glycol (PE-b-PEG) were dissolved in 0.5–5 mL of chloroform solution. The solution was then added to 5.0–30 mL of sodium hydroxide solution with a concentration of 0.001–0.008 mg / mL. The mixture was sonicated for 10–30 min at 300–600 W, followed by magnetic stirring for 20–50 min. The chloroform was removed by evaporation at 40–60 °C. The solution was then centrifuged at 12000–20000 r / min for 10–15 min, washed three times with PBS (pH 7.4), and the precipitate was dispersed in 0.5–5.0 mL of PBS buffer (pH 7.4) to obtain the hydrolyzed CUR@PSI. OAm Hydrolyzed solution;

[0019] a-2, 5-20 mg PSI OAm Dissolve the precipitate in 0.5–5 mL of chloroform solution. Then add the solution to 5.0–30 mL of sodium hydroxide solution with a concentration of 0.001–0.008 mg / mL. Sonicate for 10–30 min at 300–600 W, then magnetically stir for 20–50 min. Evaporate the chloroform at 40–60 °C. Centrifuge at 12000–20000 r / min for 10–15 min. Wash three times with PBS (pH 7.4). Disperse the precipitate in 0.5–5.0 mL of PBS buffer (pH 7.4) to obtain the hydrolyzed PSI. OAm Hydrolyzed solution, used to prepare immunogen for immunization of rabbits, and to prepare anti-PSI. OAm Antibody.

[0020] Step b specifically includes the following steps:

[0021] b-1, Take CUR@PSI from step a-1 OAm Add 0.5–5.0 mL of hydrolysis solution to 0.5–5.0 mL of PBS buffer containing 0.05–5.0 mg of N-hydroxysuccinimide (NHS) and 0.05–5.0 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride. Mix in the dark for 10–30 min, allow to stand for 30–60 min, then centrifuge. Wash three times with PBS (pH 7.4). Add 0.5–20 mg of ovalbumin (OVA) and incubate at 25°C for 2–4 h. Centrifuge at 12000–20000 rpm for 10–15 min. Disperse the precipitate in 0.5–5.0 mL of PBS (pH 7.4). Transfer the solution to a dialysis bag and dialyze in PBS buffer for at least 12 hours to obtain CUR@PSI. OAm -OVA-coated antigen;

[0022] b-2, Take the PSI from step a-2 OAm Add 0.5–5.0 mL of hydrolysis solution to 0.5–5.0 mL of PBS buffer containing 0.05–5.0 mg of N-hydroxysuccinimide (NHS) and 0.05–5.0 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride. Mix in the dark for 10–30 min, allow to stand for 30–60 min, then centrifuge. Wash three times with PBS (pH 7.4). Add 0.5–20 mg of bovine serum albumin and incubate at 25°C for 2–4 h. Centrifuge at 12000–20000 rpm for 10–15 min. Disperse the precipitate in 0.5–5.0 mL of PBS (pH 7.4). Transfer the solution to a dialysis bag and dialyze in PBS buffer for at least 12 hours to obtain PSI. OAm Immunogen solution;

[0023] In step b-1, the molecular weight cutoff of the dialysis bag is 8000-25000 Da;

[0024] Step c specifically includes the following steps:

[0025] c-1, Initial immunization: PSI OAm After mixing the immunogen solution and Freund's complete adjuvant in an equal volume ratio, the mixture was injected into the rabbits via subcutaneous injection at multiple points on the back, with 8-10 injection sites and an injection volume of 1-2 mL per rabbit. A booster immunization was administered three weeks after the initial immunization.

[0026] c-2, Enhanced Immunization: PSI OAm The immunogen and Freund's incomplete adjuvant were mixed in an equal volume ratio and injected into rabbits using the same method at 8-10 injection sites, with a dosage of 1-2 mL per rabbit. Booster immunizations were then administered every two weeks, with blood collected from the ear vein during the intervening week to measure serum titer until a titer of 1:64000 was achieved. A final booster immunization was then administered, and blood was collected from the carotid artery one week after immunization. The antiserum was allowed to settle and purified to obtain the anti-PSI drug. OAm Antibody.

[0027] Step d specifically includes the following steps:

[0028] d-1. First, under light-protected conditions, react 10–50 mg of fluorescein isothiocyanate (FITC) with 10–80 μL of 3-aminopropyltriethoxysilane (APTES) in 0.5–5.0 mL of ethanol for 24 h to obtain fluorescein isothiocyanate-labeled 3-aminopropyltriethoxysilane (FITC-APTES); store the pre-prepared FITC-APTES in a refrigerator for later use.

[0029] d-2. Quickly add 1–5 mL of 0.2% H₂PtCl₆·6H₂O solution to 20–80 mL of boiling deionized water and maintain the reaction for 30–90 s. Then, add 0.5–5.0 mL of 0.05% citric acid solution and 1% sodium citrate solution to the above solution and boil for 20–90 s. Next, add 0.5–5.0 mL of a solution containing 0.08% sodium borohydride, 1% sodium citrate, and 0.05% citric acid and boil for 5–30 min. After cooling to room temperature, a 4–6 nm Pt NPs seed solution is obtained. Add 0.5–5.0 mL of Pt The NPs seed solution was added to 20–50 mL of deionized water, followed by 0.5–5.0 mL of 0.01–0.05 M H₂PtCl₆·6H₂O solution, 0.5–5.0 mL of 1.25% ascorbic acid and 1% sodium citrate, and stirred at room temperature for 5–30 min. The reaction system was then heated to boiling and maintained for 20–60 min. After cooling to room temperature, a Pt NPs solution with a size of 25 nm and a concentration of 0.3–1.0 pM was obtained.

[0030] d-3. At room temperature, add hexadecyltrimethylammonium bromide (CTAB) aqueous solution to a 0.3–1.0 pM Pt NPs solution with a size of 25 nm, so that the concentration of CTAB in the solution is 0.001–0.01 M, and gently stir at room temperature for 10–30 min; then add 0.1–1.0 mL of ammonia water to the above solution with continuous stirring; after stirring for 10–30 min, simultaneously add 0.01–0.1 mL of tetraethyl orthosilicate (TEOS) and 0.05–0.5 mL of 20 mg / mL FITC-APTES solution, stir overnight, and then centrifuge and wash the product three times to obtain the dual-mode probe FITC@Pt Janus NPs solution.

[0031] Step d synthesized FITC@Pt Janus NPs with a particle size of 150–200 nm and uniform size distribution. Using CTAB as a template agent and TEOS as a silica precursor, CTAB and ammonia were sequentially added to a mixed solution containing Pt NPs. The mixture was slowly stirred at room temperature, followed by the simultaneous addition of TEOS and FITC-APTES. This process resulted in the slow, homogeneous nucleation and growth of fluorescent silica on the surface of the Pt NPs. FITC@Pt Janus NPs consist of two components: Pt NPs and fluorescent silica. In this invention, the aminated fluorescent dye FITC has its amino groups bonded to the hydroxyl groups of silica via hydrogen bonds, improving the photobleaching resistance of the fluorescent dye and resulting in lower background interference. Furthermore, the hydrophobic FITC binds to silica, increasing the stability of this dual-mode probe in aqueous solution. The fluorescent properties of FITC and the enzymatic catalytic function of Pt NPs are integrated into a single unit, significantly expanding the application of Janus dual-mode probes in biomarker detection. By controlling the amount of the added template agent CTAB, TEOS is homogeneously nucleated and grown into 150-200 nm silica on the surface of Pt NPs; and FITC-APTES, added simultaneously with TEOS, combines with the silanol groups of silica to obtain fluorescent silica. Thus, a dual-component system of Pt NPs and fluorescent silica is simultaneously obtained.

[0032] Step e specifically includes the following steps:

[0033] e-1, PSI resistance OAm Dissolve 0.5–5.0 mg of antibody in 0.5–5.0 mL of PBS buffer.

[0034] e-2. Take 0.5–5.0 mL of the dual-mode probe FITC@Pt Janus NPs solution, add the antibody treated in step e-1, stir at room temperature in the dark for 4–8 hours, and wash three times with PBS buffer to obtain the anti-PSI labeled with the dual-mode probe FITC@Pt Janus NPs. OAm Antibodies, for later use.

[0035] Step e synthesized FITC@Pt Janus NPs-labeled anti-PSI agents. OAm The silica component of the antibody-dual-mode probe has partially exposed amino groups on its surface, which will preferentially bind to anti-PSI antibodies. OAm The antibody's carboxyl group binds to form a peptide bond, which to some extent prevents the antibody from masking the Pt catalytic site; and the dual-mode probe and anti-PSI... OAm Antibody conjugation enhances the performance of this material on oleylamine-grafted polysuccinimide (CUR@PSI) nanocarriers loaded with the hydrophobic anticancer drug curcumin (CUR). OAm Targeted detection.

[0036] Step f specifically includes the following steps:

[0037] f-1. After the cells have grown to the logarithmic growth phase, HeLa cells are divided into groups of 5 × 10⁻⁶ cells. 4 Seeds were seeded at a density of 1 mL / mL into a laser confocal microplate and cultured until adherent.

[0038] f-2. Remove the old culture medium, wash the culture dish with PBS buffer, and add a mixture of CUR@PSI. OAm RPMI-1640 basal medium containing hydrolysate, with CUR@PSI OAm The concentration is 200–400 μg / mL. After co-culturing with cells for 30–90 min, the culture medium is aspirated and the cells are washed with PBS buffer. Then, 100 μL of cell-specific lysis buffer is added to lyse the cells, and the cell lysis buffer is collected. The cells are then treated with CUR@PSI. OAm The content is the original concentration, which can be diluted to different concentrations of CUR@PSI as needed. OAm Cell lysis buffer standard.

[0039] Step g specifically includes the following steps:

[0040] g-1, Coating: Coat CUR@PSI with carbonate buffer CB. OAm - Dilute the OVA-coated antigen to 15 μg / mL, coat 96 wells with 100 μL per well, and incubate overnight at 4°C.

[0041] g-2. Blocking: Wash three times with PBST solution, spin dry, for 3-5 minutes each time, to remove unbound CUR@PSI. OAm The antigen was coated and blocked with 200 μL of 1.5 wt% casein solution per well. The mixture was then incubated in a 37°C oven for 1–2 hours.

[0042] g-3, Sample loading competition: Wash three times with PBST solution, spin dry, 3-5 min each time, to remove excess blocking solution, then add 50 μL of the dual-mode probe FITC@Pt Janus NPs-labeled anti-PSI. OAm Antibody and 50 μL of CUR@PSI at different concentrations OAm Cell lysis buffer standards were added to each well in a gradient to induce a competitive reaction, and incubated in a 37°C oven for 2–4 hours.

[0043] g-4. Detection: Wash three times with PBST solution, spin dry, 3-5 min each time, to remove free CUR@PSI. OAmCell lysis buffer standards or antibody conjugates were used for colorimetric signal determination: 100 μL of acetate buffer, 20 μL of 2.5 mg / mT LMB, and 20 μL of 1% H2O2 solution were added to each well, and the absorbance value of each well was measured at a wavelength of 450 nm using a multi-functional microplate reader; fluorescence signal determination: the fluorescence intensity value of each well was measured at an excitation wavelength of 485 nm and an emission wavelength of 528 nm using a multi-functional microplate reader.

[0044] In step g-3, different concentrations of CUR@PSI OAm The concentrations of the cell lysis buffer standards were 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, and 100 ng / mL, respectively.

[0045] In step g-4, the dual-mode probe FITC@Pt Janus NPs serves as a marker for a dual-mode immunoassay combining colorimetry and fluorescence. The dual-mode detection results corroborate each other, resulting in higher detection accuracy. The Pt Janus NPs nanomaterial possesses peroxidase-like properties, oxidizing TMB to TMB. ox A color change occurs, generating an absorbance detection signal. FITC, as a fluorescent dye, is embedded within silica, giving FITC@Pt Janus NPs additional significant fluorescence. FITC@Pt Janus NPs can act as both an enzyme-mimicking enzyme to catalyze the oxidation of TMB to generate a colorimetric signal and a fluorescent probe to generate a fluorescence signal. The results of dual-mode immunoassay using FITC@Pt Janus NPs as markers can corroborate each other, providing a more sensitive and accurate method for the quantitative detection of the intracellular hydrophobic anticancer drug curcumin (CUR).

[0046] The linear equation of the standard curve mentioned in step h is: absorbance value A = 0.7292 - 0.1370lgC, where A is the absorbance value and C is the gradient dilution of CUR@PSI. OAm The concentration of cell lysis buffer standards and their correlation coefficient R. 2 =0.994, linear range was 0.1–100 ng / mL, detection limit was 0.0113 ng / mL. The drug content in the supernatant of curcumin nanoparticles was determined by ultraviolet spectrophotometry, and then the PSI was calculated. OAm The loading capacity for CUR was 2.22%, therefore the calculated limit of detection for CUR was 0.25 pg / mL; the fluorescence intensity value F = 15996.83 - 1536.39lgC, where F is the fluorescence intensity value and C is the CUR@PSI value. OAm CUR@PSI OAm The concentration of cell lysis buffer standards and their correlation coefficient R. 2=0.995, the linear range is 0.1~100ng / mL, the detection limit is 0.0109ng / mL, and the calculated detection limit of CUR is 0.24pg / mL. The results of the dual-mode determination are mutually corroborated, and the detection concentration is the average of the two.

[0047] This invention provides a colorimetric-fluorescence dual-mode assay for the intracellular hydrophobic anticancer drug curcumin (CUR). The method utilizes antigen-antibody specific immunoassay technology to establish a colorimetric-fluorescence dual-mode detection method. By integrating fluorescence and enzyme catalytic properties onto the same nanoparticle without interference, and using these as detection signals during the experiment, the method simultaneously performs dual-mode detection on an oleylamine-grafted polysuccinimide nanoparticle carrier loaded with the intracellular hydrophobic anticancer drug curcumin (CUR), thereby indirectly detecting the intracellular anticancer drug curcumin (CUR) with higher detection accuracy, sensitivity, and specificity.

[0048] Based on the key-lock mode of antigen-antibody one-to-one reaction, the established immunoassay method has the advantages of simple operation, high sensitivity, high specificity, high selectivity, high throughput, and non-destructive monitoring, and can be used to detect trace amounts of drugs in the human body. Enzyme-linked immunosorbent assay (ELISA) and fluorescence immunoassay (FIA) are the most commonly used immunoassay methods. Combining the two, using dual signals for ultra-trace non-destructive quantitative monitoring of drugs, will have higher accuracy.

[0049] This invention prepares curcumin (CUR@PSI) by grafting polysuccinimide with oleylamine. OAm Anti-PSI coated with antigen and labeled with dual-mode probe OAm Antibodies, specifically targeting the intracellular drug curcumin (CUR) nanocarrier oleylamine-grafted polysuccinimide (CUR@PSI) for antigen-antibody reactions. OAm Dual-mode ultra-trace detection analysis was performed to indirectly quantify the intracellular hydrophobic anticancer drug curcumin (CUR).

[0050] Compared with existing technologies, the colorimetric-fluorescence dual-mode method provided by this invention yields similar results in detecting curcumin dosage, proving that the detection results of this dual-mode method can corroborate each other. Moreover, it does not require pretreatment processes such as sample enrichment, is simple to operate, and has a short detection time. It features high throughput, high sensitivity, high specificity, precise targeting, and non-destructive detection, and can be used for ultra-trace non-destructive detection and analysis of curcumin (CUR), an intracellular hydrophobic anticancer drug. Attached Figure Description

[0051] Figure 1 TEM image of the prepared dual-mode probe FITC@Pt Janus NPs;

[0052] Figure 2 To prepare a dual-mode probe, FITC@Pt Janus NPs-labeled anti-PSI OAm TEM electron micrograph of the antibody;

[0053] Figure 3 The probe optimization curve is plotted with the amount of FITC-APTES added during the preparation of the dual-mode probe FITC@Pt Janus NPs as the x-axis and the fluorescence intensity value as the y-axis. Detailed Implementation

[0054] All reagents used can be purchased from manufacturers in the market.

[0055] The methods for preparing the solutions involved in this invention are as follows:

[0056] PBS solution (0.01 mol / L, pH = 7.4): Weigh out 8.0 g NaCl, 0.1 g KCl, and NaH2PO4·2H2O.

[0057] Dissolve 0.106g of Na2HPO4·12H2O and 3.34g of Na2HPO4·12H2O in distilled water and bring the volume to 1000mL.

[0058] Carbonate buffer CB (0.05 mol / L pH = 9.6): Weigh 1.59 g of Na2CO3 and 2.94 g of NaHCO3, dissolve them in distilled water, and bring the volume to 100 mL.

[0059] PBST solution (0.01 mol / L pH = 7.4): Add 500 μL of Tween-20 to 1000 mL of PBS and mix well.

[0060] Acetate buffer solution: Weigh 84.25g of sodium acetate, dissolve it in water, add 100ml of acetic acid, and dilute with water to 2500mL to obtain an acetate buffer solution (pH=4.2).

[0061] 1.5wt% casein solution: This is a blocking solution. Weigh 0.15g of casein and dissolve it in 10mL of PBS, then mix thoroughly.

[0062] The ammonia water used in this invention is concentrated ammonia water with a mass concentration of 22-25%.

[0063] Example 1

[0064] A colorimetric-fluorescence dual-mode method for the determination of curcumin, a hydrophobic anticancer drug, includes the following steps:

[0065] a. Preparation of PSI OAm and CUR@PSI OAm Hydrolysis solution:

[0066] a-1, 6mg PSI OAm 0.3 mg curcumin (CUR) and 0.2 mg polyethylene-b-polyethylene glycol (PE-b-PEG) were dissolved in 1.0 mL of chloroform solution. The solution was then added to 10 mL of 0.005 mg / mL sodium hydroxide solution. The mixture was sonicated for 15 min at 500 W, followed by magnetic stirring for 30 min. The chloroform was removed by evaporation at 40–60 °C. The solution was centrifuged at 12000 rpm for 10 min and washed three times with PBS solution at pH 7.4. The precipitate was then dispersed in 1 mL of PBS buffer at pH 7.4 to obtain the hydrolyzed CUR@PSI. OAm Solution;

[0067] a-2, 6mg PSI OAm Dissolve the precipitate in 1.0 mL of chloroform solution. Then add the solution to 10 mL of 0.005 mg / mL sodium hydroxide solution. Sonicate for 15 min at 500 W, then magnetically stir for 30 min. Evaporate the chloroform at 40–60 °C. Centrifuge at 12000 rpm for 10 min. Wash three times with PBS (pH 7.4). Disperse the precipitate in 1.0 mL of PBS buffer (pH 7.4) to obtain the hydrolyzed PSI. OAm Hydrolyzed solution.

[0068] b. Preparation of CUR@PSI OAm Coating antigens and immunogens:

[0069] b-1, Take CUR@PSI from step a-1 OAm 1.0 mL of hydrolysis solution was added to 1.0 mL of PBS buffer, which contained 0.35 mg of N-hydroxysuccinimide (NHS) and 0.7 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride. The mixture was stirred in the dark for 30 min, allowed to stand for 30 min, and then centrifuged. The mixture was washed three times with PBS (pH 7.4). Then, 2 mg of ovalbumin (OVA) was added, and the mixture was incubated at 25°C for 4 h. Afterward, the mixture was centrifuged at 12000 rpm for 10 min. The precipitate was dispersed in 1.0 mL of PBS (pH 7.4) buffer. The solution was then placed in a dialysis bag with a molecular weight cutoff of 8000 Da and dialyzed in PBS buffer for at least 12 hours to obtain CUR@PSI. OAm Coating antigen solution;

[0070] b-2, Take the PSI from step a-2 OAm1.0 mL of hydrolysis solution was added to 1.0 mL of PBS buffer, which contained 0.35 mg of N-hydroxysuccinimide (NHS) and 0.7 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride. The mixture was stirred in the dark for 30 min, allowed to stand for 30 min, and then centrifuged. The mixture was washed three times with PBS (pH 7.4). Then, 2 mg of bovine serum albumin was added, and the mixture was incubated at 25°C for 4 h. Afterward, the mixture was centrifuged at 12000 rpm for 10 min. The precipitate was dispersed in 1.0 mL of PBS (pH 7.4) buffer. The solution was then placed in a dialysis bag with a molecular weight cutoff of 8000 Da and dialyzed in PBS buffer for at least 12 hours to obtain CUR@PSI. OAm Immunogen solution.

[0071] c. PSI resistance OAm Antibody preparation:

[0072] c-1, Initial immunization: PSI OAm After mixing the immunogen solution and Freund's complete adjuvant in an equal volume ratio, the mixture was injected into the rabbits via subcutaneous injection at multiple points on the back, with 8-10 injection sites and an injection volume of 1-2 mL per rabbit. A booster immunization was administered three weeks after the initial immunization.

[0073] c-2, Enhanced Immunization: PSI OAm The immunogen and Freund's incomplete adjuvant were mixed in an equal volume ratio and injected into rabbits using the same method at 8-10 injection sites, with a dosage of 1-2 mL per rabbit. Booster immunizations were then administered every two weeks, with blood collected from the ear vein during the intervening week to measure serum titer until a titer of 1:64000 was achieved. A final booster immunization was then administered, and blood was collected from the carotid artery one week after immunization. The antiserum was allowed to settle and purified to obtain the anti-PSI drug. OAm Antibody.

[0074] d. Preparation of dual-mode probes FITC@Pt Janus NPs:

[0075] d-1. Under light-protected conditions, 10 mg of fluorescein isothiocyanate (FITC) was reacted with 40 μL of 3-aminopropyltriethoxysilane (APTES) in 0.5 mL of ethanol for 24 h to obtain FITC-labeled 3-aminopropyltriethoxysilane (FITC-APTES), with a concentration of 20 mg / mL. The pre-prepared FITC-APTES was stored in a refrigerator for later use.

[0076] d-2. Rapidly add 3.88 mL of 0.2% H₂PtCl₆·6H₂O solution to 50 mL of boiling deionized water and maintain the reaction for 60 s. Then, add 1.18 mL of 0.05% citric acid solution and 1% sodium citrate solution to the above solution and boil for 30 s. Next, add 0.59 mL of a solution containing 0.08% sodium borohydride, 1% sodium citrate, and 0.05% citric acid and boil for 10 min. After cooling to room temperature, a seed solution of Pt NPs with a particle size of approximately 5 nm is obtained. Add 1.0 mL of the 5 nm Pt seed to 29 mL of deionized water, then add 0.576 mL of 0.0193 M H₂PtCl₆·6H₂O solution, 0.5 mL of 1.25% ascorbic acid, and 1% sodium citrate, and stir at room temperature for 10 min. Then, heat the reaction system to boiling and maintain the boiling state for 30 min. After cooling to room temperature, a 25 nm Pt NPs solution was obtained, and the theoretically calculated concentration of 25 nm Pt NPs was 0.44 pM.

[0077] d-3. At room temperature, a 0.44 pM solution of 25 nm Pt NPs was added to a hexadecyltrimethylammonium bromide (CTAB) aqueous solution to bring the CTAB concentration to 0.004 M. The solution was then gently stirred at room temperature for 15 min. Then, 0.45 mL of ammonia was added to the solution with continuous stirring. After stirring for 10 min, 0.025 mL of tetraethyl orthosilicate (TEOS) and 0.1 mL of 20 mg / mL FITC-APTES solution were added dropwise. The mixture was stirred overnight. Subsequently, the product was centrifuged and washed three times to obtain a dual-mode probe solution of FITC@Pt Janus NPs.

[0078] Figure 1 The image shows the morphology of the dual-mode probe FITC@Pt Janus NPs. The black part represents the Pt NPs component, and the gray transparent spherical part represents the silica component embedded with FITC. The TEM electron microscopy image shows that the prepared probe has a dual-component structure. Figure 2 To combine the dual-mode probe FITC@Pt Janus NPs with PSI-resistant OAm TEM image of the antibody after successful conjugation, characterizing the dual-mode probe FITC@Pt Janus NPs and anti-PSI. OAm Antibody conjugation was successful.

[0079] e. Dual-mode probe FITC@Pt Janus NPs-labeled anti-PSI OAm Antibody preparation:

[0080] e-1, PSI resistance OAmDissolve 1 mg of antibody in 1 mL of PBS solution;

[0081] e-2. Take 1 mL of the dual-mode probe FITC@Pt Janus NPs solution prepared in step d-3, slowly add the antibody treated in step e-1, stir at room temperature in the dark for 4 h, wash three times with PBS, and you will get the Janus dual-mode probe-labeled anti-PSI. OAm Antibodies, for later use.

[0082] f、CUR@PSI OAm Preparation of cell lysate after incubation with HeLa cells:

[0083] f-1. After the cells have grown to the logarithmic growth phase, HeLa cells are divided into groups of 5 × 10⁻⁶ cells. 4 Seeds were seeded at a density of 1 mL / mL into a laser confocal microplate and cultured until adherent.

[0084] f-2. Remove the old culture medium, wash the culture dish several times with PBS, and add a mixture of CUR@PSI. OAm RPMI-1640 basal medium, in which CUR@PSI Oam The concentration was 200 μg / mL. The cells were co-cultured for 60 min, the culture medium was aspirated and washed with PBS, and then cell lysis buffer RIPA was added to lyse the cells and the cell lysis buffer was collected.

[0085] g. CUR@PSI OAm The coated antigen was diluted with coating buffer and coated into 96-well plates. After blocking, different concentrations of CUR@PSI were added. OAm Cell lysis buffer standards and dual-mode probe FITC@Pt Janus NPs-labeled anti-PSI OAm Antibody, using a dual-mode probe with dual signaling molecules, establishes a direct competitive immunoassay for the intracellular hydrophobic anticancer drug CUR@PSI. OAm :

[0086] g-1, Coating: Coat CUR@PSI with 0.05M, pH=9.6 carbonate buffer CB. OAm - Dilute the OVA-coated antigen to 15 μg / mL, coat 96 wells with 100 μL per well, and incubate overnight at 4°C.

[0087] g-2, Blocking: Remove the 96-well plate, wash three times with PBST solution, spin dry, 3 min each time, to remove unbound CUR@PSI. OAm The antigen was coated and blocked with 1.5 wt% casein solution, 200 μL per well, and incubated in an oven at 37°C for 1 h.

[0088] g-3, Sample loading competition: Wash three times with PBST solution, spin dry, 3 min each time, to remove excess blocking solution, then add 50 μL of the dual-mode probe FITC@Pt Janus NPs-labeled anti-PSI. OAm Antibody and 50 μL of CUR@PSI at concentrations of 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, and 100 ng / mL, respectively. OAm Cell lysis buffer standards were added to each well of a 96-well plate, with each concentration gradient repeated three times; different concentrations of CUR@PSI were also added. OAm The method for preparing cell lysis buffer standards is as follows: CUR@PSI is prepared by mixing CUR@PSI with 0.01 mol / L pH 7.4 PBS buffer. OAm The cell lysis buffer was diluted to the specified standard concentration; a competitive reaction was then initiated, and the cells were incubated at 37°C for 2.5 hours.

[0089] g-4. Wash three times with PBST solution, spin dry for 3 minutes each time to remove free CUR@PSI. OAm Cell lysis buffer standard or antibody conjugate, 1) Colorimetric signal determination: Add 100 μL of acetate buffer, 20 μL of 2.5 mg / mL TMB and 20 μL of 1% H2O2 solution to each well, and measure the absorbance value of each well at a wavelength of 450 nm using a multi-functional microplate reader; 2) Fluorescence signal determination: Measure the fluorescence intensity value of each well at an excitation wavelength of 485 nm and an emission wavelength of 528 nm using a multi-functional microplate reader.

[0090] CUR@PSI OAm The coated antigen, after being diluted with coating buffer, was coated onto 96-well plates. The plates were then blocked, and different concentrations of CUR@PSI were added. OAm Cell lysis buffer standard, labeled with the dual-mode probe FITC@Pt Janus NPs, containing anti-PSI OAm Antibodies were used as primary antibodies to establish a direct competitive dual-mode immunoassay for the quantitative detection of intracellular drug CUR@PSI. OAm The dual-mode probe FITC@PtJanus NPs serves as a marker for a dual-mode immunoassay combining colorimetry and fluorescence. The dual-mode detection results corroborate each other, resulting in higher detection accuracy. PtJanus NPs nanomaterials possess peroxidase-mimicking properties, oxidizing TMB to TMB. oxThe color change generates an absorbance detection signal. FITC, as a fluorescent dye, is embedded within silica, giving FITC@PtJanus NPs additional significant fluorescence. FITC@PtJanus NPs can act as both a mimic enzyme to oxidize TMB and generate a colorimetric signal, and as a fluorescent probe to generate a fluorescence signal. Using FITC@PtJanus NPs as a marker, and combining the specificity of antigen-antibody reactions, a colorimetric-fluorescence dual-mode method for the intracellular hydrophobic anticancer drug curcumin (CUR) was established. This method indirectly detects intracellular curcumin (CUR) by detecting the intracellular drug carrier oleylamine-grafted polysuccinimide loaded with the hydrophobic anticancer drug CUR. The detection results of this method are mutually corroborative, and no sample enrichment or other pretreatment processes are required. It is simple to operate, has a short detection time, and features high throughput, high sensitivity, high specificity, precise targeting, and non-destructive detection. It can perform ultra-trace, non-destructive detection and analysis of intracellular hydrophobic anticancer drugs.

[0091] h, with CUR@PSI OAm A standard curve was plotted with the logarithm of the concentration of cell lysis buffer standards on the x-axis and the absorbance values ​​on the y-axis. The linear equation of the standard curve was A = 0.7292 - 0.1370lgC, where A is the absorbance value and C is the CUR@PSI value. OAm The concentration of the cell lysis buffer standard. Its correlation coefficient R. 2 =0.994, linear range was 0.1–100 ng / mL, detection limit was 0.0113 ng / mL, and the detection limit of CUR was 0.25 pg / mL; using CUR@PSI OAm A standard curve was plotted with the logarithm of the concentration of cell lysis buffer standards on the x-axis and the fluorescence intensity value on the y-axis. The linear equation of the standard curve was F = 15996.83 - 1536.39lgC, where F is the fluorescence intensity value and C is the CUR@PSI value. OAm The concentration of the lysis buffer standard. Its R 2 =0.995, the linear range is 0.1~100ng / mL, the detection limit is 0.0109ng / mL, and the detection limit of CUR is 0.24pg / mL. The results of dual-mode detection corroborate each other.

[0092] Example 2

[0093] Detection of unknown concentrations of CUR@PSI using the colorimetric-fluorescence dual-mode assay of curcumin, a hydrophobic anticancer drug, as described in Example 1. OAm Cell lysis buffer concentration in test solution:

[0094] Repeat the steps above in Example 1, except that different concentrations of CUR@PSI from step g-3 are used. OAmThe cell lysis buffer standard was replaced with an unknown concentration of CUR@PSI. OAm Cell lysis buffer and test solution were prepared, and the absorbance at 450 nm and the fluorescence intensity at 485 nm excitation and 528 nm emission wavelengths of each well were measured using a microplate reader. Five parallel measurements were performed, and the average absorbance and fluorescence intensity were calculated. The CUR@PSI value could then be calculated based on the aforementioned standard curve. OAm The concentration of the cell lysis buffer and the test solution, and thus the binding of PSI. OAm The concentration of CUR was calculated based on its loading capacity of 2.22%. The test results are shown in Table 1.

[0095] Unknown concentration of CUR@PSI OAm The preparation method for the cell lysis buffer is as follows: CUR@PSI is prepared by mixing the lysate with 0.01 mol / L pH 7.4 PBS buffer. OAm Cell lysates were diluted at different ratios.

[0096] For spiked recovery determination, simply add 50 μL of CUR@PSI to each well in step g-3. OAm Instead of adding cell lysis buffer and test solution, add 25 μL of CUR@PSI to each well. OAm Cell lysis buffer, test solution, and 25 μL of spiked CUR@PSI OAm Cell lysis buffer, other conditions unchanged. Recovery rate = (spiked value - spiked value at 0 spiking) / spike concentration × 100%.

[0097] Table 1 Test Results

[0098]

[0099] In this experiment, the recovery rate of spiked detection in colorimetric mode was 94.8%-101.6%, and the recovery rate of spiked detection in fluorescence mode was 90.0%-101.6%. Each value was measured 5 times, and the relative standard deviations were very small.

[0100] Example 3

[0101] Investigating the amount of FITC-APTES added during the preparation of the dual-mode probe FITC@Pt Janus NPs:

[0102] Repeat the steps above in Example 1, except that the 50 μL of the dual-mode probe FITC@Pt Janus NPs labeled with anti-PSI in step g-3 is used instead. OAm Antibody and 50 μL of CUR@PSI at concentrations of 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, and 100 ng / mL, respectively. OAmThe cell lysis buffer standard was replaced with 100 μL of FITC@Pt JanusNPs-labeled anti-PSI dual-mode probe. OAm Antibody fluorescence intensity was measured in each well using a multi-functional microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 528 nm.

[0103] The amount of FITC-APTES added during the preparation of the dual-mode probe FITC@Pt Janus NPs in step d-3 was changed to 50, 60, 80, 100, and 120 μL, respectively. A probe optimization curve was plotted with the amount of FITC-APTES added during the preparation of the dual-mode probe FITC@Pt Janus NPs in step d-3 as the x-axis and the fluorescence intensity value as the y-axis. The resulting curve is shown in the figure. Figure 3 As shown, when the amount of FITC-APTES added was 100 μL, the fluorescence intensity value reached the required range (10,000–1,000,000).

[0104] FITC, as a fluorescent dye, is embedded within silica, giving FITC@Pt Janus NPs additional significant fluorescence. When the amount of embedded FITC is too small, the fluorescence intensity is too low, making it unsuitable for detecting trace drug levels. As the amount of added FITC gradually increases, the measured fluorescence intensity also increases. When the amount of added FITC reaches 100 μL, the measured fluorescence intensity reaches the required experimental range (10,000–1,000,000). Furthermore, further increasing the amount of FITC shows little change in the measured fluorescence intensity, demonstrating that when the amount of added FITC reaches 100 μL, FITC reacts sufficiently with silica, achieving the optimal fluorescence intensity.

[0105] The above method is the optimal experimental method after multiple experimental verifications. The standard curve obtained by this method has the best linear relationship and the widest linear range.

[0106] This invention has the following characteristics:

[0107] (i) A simple and green method was used to prepare FITC@Pt Janus NPs dual-mode probes as "colorimetric-fluorescence" dual-signal molecules, laying the foundation for establishing dual-mode ultra-trace detection of the intracellular hydrophobic anticancer drug curcumin (CUR);

[0108] (ii) The dual-mode probe FITC@Pt Janus NPs is loaded with fluorescent dye, which integrates the fluorescence properties of FITC and the enzyme catalytic function of PtNPs into a whole. This improves the photobleaching resistance of organic dyes, reduces background interference, enhances the stability of the probe itself in aqueous solution, and improves the sensitivity of the experiment.

[0109] (iii) The partially exposed amino groups on the silica component surface of the dual-mode probe FITC@Pt Janus NPs will preferentially interact with PSI-resistant components. OAm The antibody's carboxyl group binds to form a peptide bond, which to some extent prevents the antibody from masking the Pt catalytic site; and the dual-mode probe and anti-PSI... OAm Antibody conjugation enhances the performance of this material on oleylamine-grafted polysuccinimide (CUR@PSI) nanocarriers loaded with the hydrophobic anticancer drug curcumin (CUR). OAm Targeted detection;

[0110] (iv) A colorimetric-fluorescence dual-mode assay for the indirect detection of curcumin (CUR), a hydrophobic anticancer drug in cells, was established by utilizing the specificity of the dual-mode probe FITC@Pt Janus NPs in the reaction with antigens and antibodies. The dual-mode detection results corroborate each other, providing a rapid and accurate quantitative detection method for ultra-trace non-destructive quantitative detection of curcumin (CUR) in cells in the future.

[0111] (v) This method is simple, fast, accurate, highly sensitive and specific, and can realize the dual-channel sensitive and accurate ultra-trace non-destructive quantitative detection of curcumin (CUR), an intracellular hydrophobic anticancer drug.

[0112] The above detailed description of the method for determining intracellular nanodrug curcumin (CUR) using a colorimetric-fluorescence dual-mode immunosensor based on dual-mode probe labeling, as described in the above-described embodiments, is illustrative rather than limiting. Several embodiments may be listed within the defined scope. Therefore, variations and modifications that do not depart from the overall concept of the present invention should be within the scope of protection of the present invention.

Claims

1. A colorimetric-fluorescence dual-mode determination method for curcumin, a hydrophobic anticancer drug, characterized in that, The colorimetric-fluorescence dual-mode determination method includes the following steps: a. Preparation of nanomedicine carrier, oleamine dendron-grafted polysuccinimide PSI OAm and oleamine dendron-grafted polysuccinimide loaded curcumin CUR@PSI OAm hydrolysis solution; b. Preparation of CUR @PSI OAm Coating antigens and immunogens; c. Anti-PSI OAm Preparation of antibodies; d. Preparation of dual-mode probes FITC@Pt Janus NPs; e. Dual-mode probe FITC@Pt Janus NPs labeled anti-PSI OAm Preparation of antibodies; f、CUR @PSI OAm Preparation of cell lysate after incubation with HeLa cells; g. CUR @PSI OAm The coating antigen was diluted with coating buffer and then coated into 96-well plates. The plates were then blocked, and lysis buffer standards of different concentrations and anti-PSI labeled with a dual-mode probe were added. OAm Antibody, using colorimetric and fluorescent dual-signal molecules with dual-mode probes, to establish a direct competitive dual-mode immunoassay for the intracellular hydrophobic anticancer drug CUR@PSI OAm ; h, with CUR @PSI OAm A standard curve was plotted with the logarithm of the concentration of the lysis buffer standard on the x-axis and the absorbance or fluorescence intensity on the y-axis. This curve was obtained by grafting polysuccinimide (CUR@PSI) onto an oleylamine nanocarrier loaded with the anticancer drug curcumin (CUR) into cells. OAm The detection of curcumin (CUR), a hydrophobic anticancer drug, was indirectly performed on cells. Step d specifically includes the following steps: d-1. First, under light-protected conditions, 10-50 mg of fluorescein isothiocyanate (FITC) was reacted with 10-80 µL of 3-aminopropyltriethoxysilane (APTES) in 0.5-5.0 mL of ethanol for 24 h to obtain fluorescein isothiocyanate-labeled 3-aminopropyltriethoxysilane (FITC-APTES); the pre-prepared FITC-APTES was stored in a refrigerator for later use. d-2. Quickly add 1-5 mL of 0.2% H₂PtCl₆·6H₂O solution to 20-80 mL of boiling deionized water and maintain the reaction for 30-90 s. Then, add 0.5-5.0 mL of 0.05% citric acid solution and 1% sodium citrate solution to the above solution and boil for 20-90 s. Next, add 0.5-5.0 mL of a solution containing 0.08% sodium borohydride, 1% sodium citrate, and 0.05% citric acid and boil for 5-30 min. After cooling to room temperature, a 4-6 nm Pt NPs seed solution is obtained. Add 0.5-5.0 mL of the Pt NPs seed solution to 20-50 mL of deionized water, then add 0.5-5.0 mL of 0.01-0.05 M H₂PtCl₆·6H₂O solution and 0.5-5.0 mL of [other ingredients not specified in the original text]. 1.25% ascorbic acid and 1% sodium citrate were added and stirred at room temperature for 5-30 min; then the reaction system was heated to boiling and maintained for 20-60 min; cooled to room temperature to obtain a Pt NPs solution with a size of 25 nm. d-3. At room temperature, add hexadecyltrimethylammonium bromide (CTAB) aqueous solution to a 0.3-1.0 pM solution of 25 nm Pt NPs, so that the concentration of CTAB in the solution is 0.001-0.01 M, and stir gently at room temperature for 10-30 min. Then, add 0.1-1.0 mL of ammonia water to the above solution with continuous stirring. After stirring for 10-30 min, add 0.01-0.1 mL of tetraethyl orthosilicate (TEOS) and 0.05-0.5 mL of 20 mg / mL FITC-APTES solution dropwise. Stir overnight. Then, centrifuge and wash the product three times to obtain the dual-mode probe FITC@Pt Janus NPs solution.

2. The colorimetric-fluorescence dual-mode determination method according to claim 1, characterized in that, Step a specifically includes the following steps: a-1, 5-20 mg PSI OAm 0.2–5 mg curcumin (CUR) and 0.2–5 mg polyethylene-b-polyethylene glycol (PE-b-PEG) were dissolved in 0.5–5 mL of chloroform solution. The solution was then added to 5.0–30 mL of sodium hydroxide solution with a concentration of 0.001–0.008 mg / mL. The mixture was sonicated for 10–30 min at 300–600 W, followed by magnetic stirring for 20–50 min. The chloroform was removed by evaporation at 40–60 °C. The solution was then centrifuged at 12000–20000 r / min for 10–15 min, washed three times with PBS (pH 7.4), and the precipitate was dispersed in 0.5–5.0 mL of PBS buffer (pH 7.4) to obtain the hydrolyzed CUR@PSI. OAm Hydrolyzed solution; a-2, 5-20 mg PSI OAm Dissolve the precipitate in 0.5–5 mL of chloroform solution. Then add the solution to 5.0–30 mL of sodium hydroxide solution with a concentration of 0.001–0.008 mg / mL. Sonicate for 10–30 min at 300–600 W, then magnetically stir for 20–50 min. Evaporate the chloroform at 40–60 °C. Centrifuge at 12000–20000 r / min for 10–15 min. Wash three times with PBS (pH 7.4). Disperse the precipitate in 0.5–5.0 mL of PBS buffer (pH 7.4) to obtain the hydrolyzed PSI. OAm Hydrolyzed solution.

3. The colorimetric-fluorescence dual-mode determination method according to claim 1, characterized in that, Step b: Preparation of CUR@PSI OAm The specific steps for coating the antigen are as follows: Take CUR@PSI from step a-1 OAm Add 0.5–5.0 mL of hydrolysis solution to 0.5–5.0 mL of PBS buffer containing 0.05–5.0 mg of N-hydroxysuccinimide (NHS) and 0.05–5.0 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride. Mix in the dark for 10–30 min, allow to stand for 30–60 min, then centrifuge. Wash three times with PBS (pH 7.4). Add 0.5–20 mg of ovalbumin (OVA) and incubate at 25°C for 2–4 h. Centrifuge at 12000–20000 r / min for 10–15 min. Disperse the precipitate in 0.5–5.0 mL of PBS (pH 7.4). Transfer the solution to a dialysis bag and dialyze in PBS buffer for at least 12 hours to obtain CUR@PSI. OAm -OVA coated antigen.

4. The colorimetric-fluorescence dual-mode determination method according to claim 1, characterized in that, Step e specifically includes the following steps: e-1, PSI resistance OAm Dissolve 0.5–5.0 mg of antibody in 0.5–5.0 mL of PBS buffer. e-2. Take 0.5 ~ 5.0 mL of the dual-mode probe FITC @Pt Janus NPs solution, add the antibody treated in step e-1, stir at room temperature in the dark for 4 ~ 8 h, and wash three times with PBS buffer to obtain the anti-PSI labeled with the dual-mode probe FITC @Pt Janus NPs. OAm Antibodies, for later use.

5. The colorimetric-fluorescence dual-mode determination method according to claim 1, characterized in that, Step f specifically includes the following steps: f-1. After the cells have grown to the logarithmic growth phase, HeLa cells are divided into groups of 5 × 10⁻⁶ cells. 4 Seeds were seeded at a density of 1 mL / mL into a laser confocal microplate and cultured until adherent. f-2. Remove the old culture medium, wash the culture dish with PBS buffer, and add a mixture of CUR@PSI. OAm RPMI-1640 basal medium containing hydrolysate, with CUR@PSI OAm The concentration is 200~400µg / mL. Co-culture with cells for 30~90 min, aspirate the culture medium and wash with PBS buffer, then add 100µL of cell-specific lysis buffer to lyse the cells and collect the cell lysis buffer.

6. The colorimetric-fluorescence dual-mode determination method according to claim 1, characterized in that, Step g specifically includes the following steps: g-1, Coating: Coat CUR @PSI with carbonate buffer CB. OAm - Dilute the OVA-coated antigen to 15 µg / mL, coat 96 wells with 100 µL per well, and incubate overnight at 4°C. g-2, Blocking: Wash three times with PBST solution, spin dry, 3-5 min each time, to remove unbound CUR@PSI. OAm The antigen was coated and blocked with 1.5 wt% casein solution, 200 µL per well, and incubated in a 37°C oven for 1 to 2 h. g-3, Sample loading competition: Wash three times with PBST solution, spin dry, 3-5 min each time, to remove excess blocking solution, then add 50 µL of dual-mode probe FITC@Pt Janus NPs-labeled anti-PSI. OAm Antibody and 50 µL of different concentrations of CUR @PSI OAm Cell lysis buffer standards were added to each well in a gradient to induce a competitive reaction, and the cells were incubated in a 37°C oven for 2 to 4 hours. g-4. Detection: Wash three times with PBST solution, spin dry, 3-5 min each time, to remove free CUR@PSI. OAm Cell lysis buffer standards or antibody conjugates were used for colorimetric signal determination: 100 µL of acetate buffer, 20 µL of 2.5 mg / mL TMB, and 20 µL of 1% H2O2 solution were added to each well, and the absorbance of each well was measured at a wavelength of 450 nm using a multi-mode microplate reader; fluorescence signal determination: the fluorescence intensity of each well was measured at an excitation wavelength of 485 nm and an emission wavelength of 528 nm using a multi-mode microplate reader.

7. The colorimetric-fluorescence dual-mode determination method according to claim 1, characterized in that, The linear equation of the standard curve mentioned in step h is absorbance value A = 0.7292 - 0.1370 lgC, where A is the absorbance value and C is the gradient dilution of CUR@PSI. OAm The concentration of cell lysis buffer standards and their correlation coefficient R. 2 =0.994, with a linear range of 0.1~100 ng / mL.

8. The colorimetric-fluorescence dual-mode determination method according to claim 1, characterized in that, The linear equation of the standard curve mentioned in step h is: fluorescence intensity value F = 15996.83 - 1536.39lgC, where F is the fluorescence intensity value and C is the gradient dilution of CUR@PSI. OAm The concentration of cell lysis buffer standards and their correlation coefficient R. 2 = 0.995, with a linear range of 0.1~100 ng / mL.

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