Preparation method of double-ligand peptide / UiO-66-NH2-coated AYG fluorescent probe

Abstract

The invention belongs to the technical field of biomedicine detection, and provides a preparation method of a double-ligand peptide / UiO-66-NH2-atAYG fluorescent probe and application of the double-ligand peptide / UiO-66-NH2-atAYG fluorescent probe in CA153 detection.The fluorescent probe has the advantages of being high in sensitivity and specificity, good in stability, low in cost, easy and convenient to operate and the like, can meet the requirement for precise detection of trace CA153, and has a good application prospect. The method is especially suitable for primary medical institutions and large-scale clinical screening.

Description

Technical Field

[0001] This application relates to the field of biomedical detection technology, specifically to a method for preparing a dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe and its application in the ultrasensitive detection of carbohydrate antigen 153 (CA153). Background Technology

[0002] Carbohydrate antigen 153 (CA153) is a specific antigenic epitope of a mucin glycoprotein encoded by the MUC-1 gene. As a marker for recurrence in stage II / III breast cancer, it is one of the core serum tumor markers most closely associated with breast cancer in clinical practice. Numerous clinical studies have confirmed that the serum CA153 concentration in breast cancer patients is significantly higher than that in patients with benign breast diseases, while the serum CA153 concentration in early-stage breast cancer patients is extremely low (only 20%-40% shows an increase). Its dynamic changes can provide crucial evidence for early diagnosis, disease progression assessment, treatment monitoring, and recurrence early warning in breast cancer. Furthermore, CA153 can also serve as an auxiliary diagnostic marker for other malignant tumors such as lung cancer, ovarian cancer, and cervical cancer, providing important references for clinical screening and differential diagnosis of related diseases.

[0003] Currently, enzyme-linked immunosorbent assay (ELISA) is the primary clinical method for detecting CA153. Other detection techniques, such as surface-enhanced Raman scattering (SERS), microfluidic biosensors, and electrochemiluminescence immunoassay, have also been reported. While these techniques possess certain detection capabilities, they generally suffer from complex procedures and require highly skilled personnel and equipment. Their practical application is limited, particularly in situations with scarce medical resources, large-scale community screening, and difficulties with invasive sampling, hindering widespread adoption. Therefore, developing fluorescent probes based on UiO-66-NH2 and dual-ligand peptides holds promise for overcoming the shortcomings of existing CA153 detection technologies, achieving ultrasensitive, low-cost, and convenient detection, and possessing significant clinical application value. Summary of the Invention

[0004] The purpose of this application is to provide a method for preparing a dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe and its application in the detection of CA153. This fluorescent probe has the advantages of high sensitivity, high specificity, good stability, low cost and simple operation, and can meet the needs of accurate detection of trace CA153, especially suitable for primary medical institutions and large-scale clinical screening.

[0005] The technical solution of this application is as follows: Firstly, this application provides a method for preparing a dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe, characterized in that the method includes:

[0006] Step 1: Mix UiO-66-NH2, dual-ligand peptide, crosslinking agent and reaction solvent, and obtain dual-ligand peptide / UiO-66-NH2 composite nanoparticles by isothermal reaction; wherein, the dual-ligand peptide includes SEQ ID NO.1: GTTFSNYW and SEQ ID NO.2: MHYLEYPF, and the crosslinking agent includes o-phthalaldehyde (OPA).

[0007] Step 2: The dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe was obtained by mixing and incubating the dual-ligand peptide / UiO-66-NH2 composite nanoparticles with the fluorescent signal molecule acridine yellow G (AYG) at room temperature.

[0008] In some embodiments, step 1 includes mixing UiO-66-NH2, GTTFSNYW, MHYLEYPF, and o-phthalaldehyde in proportion, ultrasonically dispersing for 1-3 min, purging with nitrogen for 4-6 min, and reacting in a water bath at 60-70°C under sealed conditions.

[0009] In some embodiments, the reaction solvent is one of methanol, n-butanol, dimethyl sulfoxide, and N,N-dimethylformamide, preferably dimethyl sulfoxide.

[0010] In some embodiments, the reaction solvent is one of methanol, n-butanol, dimethyl sulfoxide, and N,N-dimethylformamide, and the amount used is 20 mL (corresponding to 0.01 mmol UiO-66-NH2).

[0011] In some implementations, the reaction time is 6 to 30 hours, preferably 18 to 24 hours, and more preferably 24 hours.

[0012] In some embodiments, step 2 includes: the concentration of the dual-ligand peptide / UiO-66-NH2 composite nanoparticles is 50-500 μg / mL, the concentration of acridine yellow G is 5-15 μg / mL, the incubation time at room temperature is 20-40 min, and the reaction solvent is methanol.

[0013] In some embodiments, the molar ratio of GTTFSNYW to MHYLEYPF in the dual-ligand peptide is 1:1.

[0014] In some embodiments, the molar ratio of GTTFSNYW, MHYLEYPF, and o-phthalaldehyde is 1:1:(1-24); preferably 1:1:(12-24), and more preferably 1:1:18.

[0015] In some embodiments, the molar ratio of UiO-66-NH2, GTTFSNYW, MHYLEYPF, and phthalaldehyde is 1:1:1:(1-24), preferably 1:1:1:(12-24), and more preferably 1:1:1:18.

[0016] In a preferred embodiment of the present invention,

[0017] Step 1 includes: mixing UiO-66-NH2, GTTFSNYW, MHYLEYPF, and phthalaldehyde in a specified ratio, ultrasonically dispersing for 1-3 minutes, purging with nitrogen for 4-6 minutes, and reacting under sealed conditions in a water bath at 60-70°C. The reaction solvent is dimethyl sulfoxide, the reaction time is 24 hours, and the molar ratio of UiO-66-NH2, GTTFSNYW, MHYLEYPF, and phthalaldehyde is 1:1:1:18.

[0018] Step 2 includes: reacting the dual-ligand peptide / UiO-66-NH2 composite nanoparticles with the fluorescent signal molecule acridine yellow G (AYG) to obtain a dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe, wherein the concentration of the dual-ligand peptide / UiO-66-NH2 composite nanoparticles is 500 μg / mL, the concentration of acridine yellow G is 15 μg / mL, the mixture is mixed and incubated at room temperature for 20~40 min, and the reaction solvent is methanol.

[0019] Secondly, this application provides a dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe prepared by the method described in the first aspect.

[0020] Thirdly, this application provides the application of a dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe prepared by the method described in the first aspect in the detection of CA153.

[0021] In some implementations, the test subjects include spiked mouse serum samples or serum samples from clinical cancer patients.

[0022] In some implementations, the fluorescent probe and sample are incubated in the dark with shaking for 10-40 minutes during detection, and the adsorption equilibrium time is 10-30 minutes.

[0023] In some implementations, the fluorescent probe and sample are incubated in the dark with shaking for 30 minutes during detection, and the adsorption equilibrium time is 20 minutes.

[0024] Beneficial effects

[0025] This application uses phthalaldehyde as a crosslinking agent and a dual-ligand peptide as a specific recognition element. It utilizes the specific Schiff base reaction between phthalaldehyde and the amino groups on the surface of UiO-66-NH2 and the amino groups of the dual-ligand peptide, as well as the synergistic recognition effect of the dual-ligand peptide on different key epitopes of CA153, to improve the selectivity and stability of the recognition sites. The prepared fluorescent probe exhibits ultra-high specificity for CA153, high detection sensitivity, and fast response speed. When used as a CA153 detection tool, it can maintain a high recovery rate of CA153 in mouse serum with complex matrices and in the serum of clinical cancer patients.

[0026] The advantages of this application are: 1. This application uses dual-ligand peptides GTTFSNYW and MHYLEYPF to target different key epitopes of CA153, forming a synergistic recognition effect, which significantly improves the binding affinity and specificity of the probe to CA153, effectively resisting the influence of interfering proteins in complex serum matrix, with selectivity factors greater than 3; 2. Based on the fluorescence enhancement mechanism triggered by the binding of CA153 to the probe, the established detection system shows a good linear relationship in the concentration range of 0.05-1.5 U / mL (R0). 2 =0.998), with a detection limit as low as 0.004147 U / mL, which is 3 orders of magnitude higher than the routine clinical ELISA (1U / mL), meeting the detection requirements for trace amounts of CA153; 3. It does not require complex and expensive detection equipment, requires a small sample volume, and is suitable for primary healthcare institutions and large-scale clinical screening. It provides a new analytical tool for the early diagnosis and disease monitoring of breast cancer and other related malignant tumors, and has important practical value and translational potential. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 Scanning electron microscope image of the fluorescent probe;

[0029] Figure 2 Comparison of dual-ligand peptide / UiO-66-NH2 composite nanoparticles prepared using different proportions of crosslinking agents in the fluorescent probe preparation method provided in the embodiments of this application;

[0030] Figure 3 Comparison of dual-ligand peptide / UiO-66-NH2 composite nanoparticles prepared with different solvents in the fluorescent probe preparation method provided in the embodiments of this application;

[0031] Figure 4 Comparison of dual-ligand peptide / UiO-66-NH2 composite nanoparticles prepared using different reaction times in the fluorescent probe preparation method provided in the embodiments of this application;

[0032] Figure 5 Comparison of different concentrations of dual-ligand peptide / UiO-66-NH2 composite nanoparticles and acridine yellow G in the fluorescent probe preparation method provided in the embodiments of this application;

[0033] Figure 6 A schematic diagram of the identification mechanism;

[0034] Figure 7 The graph shows the actual recovery rate of CA153 spiked samples.

[0035] Figure 8 Brand-Altmann diagram of enzyme-linked immunosorbent assay (ELISA) and fluorescent probe in CA153 protein detection. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0037] Existing fluorescent probe technology typically consists of a recognition unit, a signal transduction unit, and a carrier unit, achieving targeted capture through the specific interaction between the recognition unit and the target molecule. Due to their excellent specificity and sensitivity, fluorescent probes have been successfully applied to the detection of target molecules in various complex matrices, such as membrane matrix metalloproteinase type 1 (MT1-MMP) in blood samples, hepatic proteinase K (CatK) in osteoclasts, protein tyrosine kinase 7 (PTK) in MCF-7 cells and human serum. However, existing fluorescent probes still have some limitations: such as poor photostability, prone to photobleaching under prolonged excitation, affecting the persistence and accuracy of detection; and complex synthesis and modification processes, resulting in high preparation costs and limiting large-scale applications. To address these issues, the introduction of nanomaterials into fluorescent probes has shown significant application potential in multiple biomedical fields, including small molecule recognition, biomolecule tracking, visualization of intracellular structures, and in vivo disease diagnosis.

[0038] To address the aforementioned issues, this application creatively constructs a dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe for the ultrasensitive quantitative detection of CA153. The experimental system optimized the probe's preparation parameters and response conditions, and its physicochemical properties, dynamic adsorption properties, detection performance, binding stability, reproducibility, and selectivity were investigated in detail. Finally, the constructed probe was used for the sensitive quantitative detection of CA153 in spiked serum samples and trace amounts of blood samples from cancer patients.

[0039] The following will describe this in conjunction with specific embodiments.

[0040] Specifically, this application provides a method for preparing a dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe, the method comprising:

[0041] S1. Mix UiO-66-NH2, dual-ligand peptides, crosslinking agents, and reaction solvents, and obtain dual-ligand peptide / UiO-66-NH2 composite nanoparticles through a isothermal reaction; wherein, the dual-ligand peptides include GTTFSNYW and MHYLEYPF, and the crosslinking agent includes o-phthalaldehyde (OPA).

[0042] S2. The dual-ligand peptide / UiO-66-NH2 composite nanoparticles are reacted with the fluorescent signal molecule acridine yellow G (AYG) to obtain the dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe.

[0043] The method for preparing the dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe provided in this application embodiment utilizes the epitope structure matching characteristics between the dual-ligand peptides (GTTFSNYW and MHYLEYPF) and CA153. The cross-linking agent phthalaldehyde forms stable imine bonds with the amino groups on the surface of UiO-66-NH2 and the amino groups of the dual-ligand peptides. Simultaneously, the dual-ligand peptides and AYG construct a signal layer through affinity, which can fix the recognition site and signal molecule. Thus, in the detection of CA153, the synergistic recognition effect of the prepared fluorescent probe not only endows the probe with specific binding ability to key epitopes of CA153, but also achieves a high-efficiency response of the fluorescence signal by competing with the specificity of AYG, thereby improving the detection sensitivity and anti-interference performance.

[0044] In some embodiments, the molar ratio of UiO-66-NH2, GTTFSNYW, MHYLEYPF, and phthalaldehyde is 1:1:1:(1-24). Optionally, the molar ratio of UiO-66-NH2, GTTFSNYW, MHYLEYPF, and phthalaldehyde can be 1:1:1:1, 1:1:1:2, 1:1:1:4, 1:1:1:12, 1:1:1:18, 1:1:1:24, or any ratio within the above range.

[0045] In some embodiments, the reaction solvent is one of methanol, n-butanol, dimethyl sulfoxide, and N,N-dimethylformamide. Preferably, the amount used is 20 mL (corresponding to 0.01 mmol UiO-66-NH2).

[0046] In some embodiments, the reaction time of the dual-ligand peptide / UiO-66-NH2 composite nanoparticles is (6-30) h. Optionally, the reaction time of the dual-ligand peptide / UiO-66-NH2 composite nanoparticles can be any ratio within the range of 6h, 7h, 8h, 9h, 10h, 11h, 12h, 18h, 24h, 30h, or above.

[0047] In some embodiments, the ligand peptide / UiO-66-NH2 composite nanoparticles are reacted with the fluorescent signaling molecule acridine yellow G (AYG) to obtain a dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe.

[0048] In some embodiments, the concentration of the ligand peptide / UiO-66-NH2 composite nanoparticles is (50-500) μg / mL. Optionally, the concentration of the ligand peptide / UiO-66-NH2 composite nanoparticles can be 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 450 μg / mL, 500 μg / mL, or any ratio within the above range.

[0049] In some embodiments, the concentration of acridine yellow G is (5-15) μg / mL. Optionally, the concentration of the dual-ligand peptide / UiO-66-NH2 composite nanoparticles can be 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 11 μg / mL, 13 μg / mL, 15 μg / mL, or any ratio within the above range.

[0050] In some embodiments, the preparation of the dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe requires a heating reaction of 30 min.

[0051] This application also provides a method for preparing a dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe as described in any of the above embodiments.

[0052] This application also provides an application of the dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe prepared by any of the above embodiments in the detection of CA513.

[0053] The method for preparing the dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe provided in this application uses phthalaldehyde as a cross-linking agent. It leverages the specific Schiff base reaction between phthalaldehyde and the amino groups on the surface of UiO-66-NH2 and the amino groups of the dual-ligand peptide molecule, as well as the affinity between the dual-ligand peptide and the fluorescent molecule AYG, to improve the density of recognition sites and the stability of the signal layer. The prepared fluorescent probe exhibits ultra-high specificity for CA153, high detection sensitivity, and fast response speed. When used as a CA153 detection tool, it can maintain a high recovery rate of CA153 in mouse serum and clinical cancer patient serum with complex matrices. The method is low-cost, convenient to operate, and the reaction conditions are mild and easily controlled. The prepared fluorescent probe can be used for trace CA153 detection, achieving ultrasensitive quantitative analysis of CA153 in complex biological matrices, and showing a strong positive correlation with the results of conventional clinical ELISA methods, providing reliable technical support for early cancer screening.

[0054] The following will describe the scheme of this application in detail with reference to the accompanying drawings and various embodiments. GTTFSNYW and MHYLEYPF were synthesized and provided by Shanghai Chutai Co., Ltd., and UiO-66-NH2 was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Z282601).

[0055] Example 1: This example provides a method for preparing dual-ligand peptide / UiO-66-NH2 composite nanoparticles, including:

[0056] 0.01 mmol of GTTFSNYW, 0.01 mmol of MHYLEYPF, 0.01 mmol of UiO-66-NH2, and 0.01 mmol of o-phthalaldehyde were added to a single-necked flask. After dissolving in 20 mL of methanol, the mixture was sonicated for 2 min to ensure homogeneity. The flask was then sealed with a sealing film at the joint and further wrapped with plastic wrap. The entire flask, from the neck down, was immersed in a water bath at 65 °C for 24 h. After the reaction, dual-ligand peptide / UiO-66-NH2 composite nanoparticles were obtained.

[0057] Example 2: This example provides a method for preparing dual-ligand peptide / UiO-66-NH2 composite nanoparticles, including:

[0058] 0.01 mmol of GTTFSNYW, 0.01 mmol of MHYLEYPF, 0.01 mmol of UiO-66-NH2, and 0.18 mmol of o-phthalaldehyde were added to a single-necked flask. After dissolving in 20 mL of methanol, the mixture was sonicated for 2 min to ensure homogeneity. The flask was then sealed with a sealing film at the joint and further wrapped with plastic wrap. The entire flask, from the neck down, was immersed in a water bath at 65 °C for 24 h. After the reaction, dual-ligand peptide / UiO-66-NH2 composite nanoparticles were obtained.

[0059] Example 3: This example provides a method for preparing a dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe, including:

[0060] 0.01 mmol of GTTFSNYW, 0.01 mmol of MHYLEYPF, 0.01 mmol of UiO-66-NH2, and 0.18 mmol of o-phthalaldehyde were added to a single-necked flask. 20 mL of dimethyl sulfoxide was added to dissolve the nanoparticles, and the mixture was sonicated for 2 min to ensure homogeneity. The flask was sealed with sealing film at the joint and then wrapped with plastic wrap. The entire flask, from the neck down, was immersed in a water bath at 65 °C for 24 h. After the reaction, dual-ligand peptide / UiO-66-NH2 composite nanoparticles were obtained. A concentration of 50 μg / mL for the dual-ligand peptide / UiO-66-NH2 composite nanoparticles and 5 μg / mL for acridine yellow G were added, and the mixture was brought to a final volume of 10 mL with methanol. After incubation at room temperature for 30 min, the dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe was obtained.

[0061] Comparative Example 1: The preparation process of the dual-ligand peptide / UiO-66-NH2 composite nanoparticles in this comparative example is the same as that in Example 1. The only difference between this example and Example 1 is the change in the amount of phthalaldehyde added.

[0062] Comparative Example 2: The preparation process of the dual-ligand peptide / UiO-66-NH2 composite nanoparticles in this comparative example is the same as that in Example 2. The only difference between the two examples is the change in the reaction solvent.

[0063] Comparative Example 3: The preparation process of the dual-ligand peptide / UiO-66-NH2 composite nanoparticles in this comparative example is the same as that in Example 2. The only difference between Example 2 and Example 2 is the change in reaction time.

[0064] Comparative Example 4: In this comparative example, a dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe was prepared. The preparation process was the same as in Example 3. The only difference from Example 3 was the change in the concentration of the dual-ligand peptide / UiO-66-NH2 composite nanoparticles and the change in the concentration of acridine yellow G.

[0065] Performance tests of the dual-ligand peptide / UiO-66-NH2 composite nanoparticles and dual-ligand peptide / UiO-66-NH2@AYG fluorescent probes prepared in the above examples and comparative examples:

[0066] Take 1 mL of the dual-ligand peptide / UiO-66-NH2 composite nanoparticle solution and use high performance liquid chromatography (HPLC) to detect the content of unimmobilized ligand peptides in the reaction solution;

[0067] Accurately prepare 10 mL of the dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe solution, and measure the fluorescence intensity in triplicate. Based on the characteristic that the dual-ligand peptide / UiO-66-NH2 composite nanoparticles quench fluorescence after adsorbing fluorescein, the relationship between the degree of quenching (ΔF / F) and the concentration of the target substance (dual-ligand peptide / UiO-66-NH2 composite nanoparticles) conforms to the Stern-Volmer equation, and the formula for its fluorescence quenching efficiency is as follows:

[0068] ∆F / F=1+KSV*C

[0069] In the formula, F represents the blank fluorescein, ΔF is the difference between the fluorescence intensity of the dual-ligand peptide / UiO-66-NH2 composite nanoparticles after adsorbing fluorescein and the blank; Ksv is the Stern-Volmer constant, representing the fluorescence quenching efficiency; [C] represents the dual-ligand peptide / UiO-66-NH2 composite nanoparticles.

[0070] Experimental results:

[0071] Appendix Figure 2 As a comparative example, the amount of ligand peptide immobilized in the solution of the dual-ligand peptide / UiO-66-NH2 composite nanoparticles prepared under different amounts of phthalaldehyde shows that under the same reaction conditions, the amount of immobilized dual-ligand peptide is the largest when the molar ratio of dual-ligand peptide to phthalaldehyde is 1:1:18.

[0072] Appendix Figure 3 The amount of ligand peptide immobilized in the solution of the dual-ligand peptide / UiO-66-NH2 composite nanoparticles prepared in Comparative Example 2 under different reaction solvents is shown to be the largest amount of ligand peptide immobilized when the reaction solvent is dimethyl sulfoxide, under the same reaction conditions.

[0073] Appendix Figure 4To compare the amount of ligand peptides immobilized in the solution of the dual-ligand peptide / UiO-66-NH2 composite nanoparticles prepared in Example 3 at different reaction times, it is shown that under the same reaction conditions, the amount of immobilized ligand peptides is the largest when the reaction time is 24 h.

[0074] Appendix Figure 5 To compare the fluorescence quenching efficiency of the dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe prepared under different concentrations of dual-ligand peptide / UiO-66-NH2 composite nanoparticles and acridine yellow G, it is shown that under the same reaction conditions, the fluorescence quenching efficiency is the highest when the concentration of dual-ligand peptide / UiO-66-NH2 composite nanoparticles is 500 μg / mL and the concentration of acridine yellow G is 15 μg / mL.

[0075] Appendix Figure 6 This is a schematic diagram of the identification mechanism.

[0076] Appendix Figure 7 For practical sample application: CA153 standard was added to the dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe solution to prepare a series of CA153 standard solutions of different concentrations. The solutions were incubated at room temperature in the dark for 20 min. The content was determined based on the change in fluorescence difference, and the recovery rate was calculated according to the standard curve.

[0077] Appendix Figure 8 To compare the CA153 detection results using ELISA and fluorescent probe methods using the Brand-Altmann plot, the mean deviation was -1.26, the SD was 5.03, and the agreement limit ranged from -11.13 to 8.60, with all data points falling within these limits. Although a slight deviation exists, it is within an acceptable range.

[0078] Table 1 shows the consistency of detection results of enzyme-linked immunosorbent assay (ELISA) and fluorescent probe in CA153 protein detection using Pearson analysis. The analysis results show that the concentration of CA153 detected by the two methods is significantly and strongly positively correlated (r=0.9944, n=10, P<0.001), indicating that the detection trends of the two methods are highly consistent.

[0079] Table 1

[0080]

[0081] The method for preparing the dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe provided in this application uses phthalaldehyde as a cross-linking agent. It leverages the specific Schiff base reaction between phthalaldehyde and the amino groups on the surface of UiO-66-NH2 and the amino groups of the dual-ligand peptide molecule, as well as the affinity between the dual-ligand peptide and the fluorescent molecule AYG, to improve the density of recognition sites and the stability of the signal layer. The prepared fluorescent probe exhibits ultra-high specificity for CA153, high detection sensitivity, and fast response speed. When used as a CA153 detection tool, it can maintain a high recovery rate of CA153 in mouse serum and clinical cancer patient serum with complex matrices. The method is low-cost, convenient to operate, and the reaction conditions are mild and easily controlled. The prepared fluorescent probe can be used for trace CA153 detection, achieving ultrasensitive quantitative analysis of CA153 in complex biological matrices, and showing a strong positive correlation with the results of conventional clinical ELISA methods, providing reliable technical support for early cancer screening.

[0082] Of course, the above embodiments are only for illustrating the technical concept and features of this application, and their purpose is to enable those skilled in the art to understand the content of this application and implement it accordingly. They should not be used to limit the scope of protection of this application. All modifications made in accordance with the spirit and essence of the main technical solution of this application should be included within the scope of protection of this application.

Claims

1. A method for preparing a dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe, characterized in that, The method includes: Step 1: Mix UiO-66-NH2, dual-ligand peptide, crosslinking agent and reaction solvent, and obtain dual-ligand peptide / UiO-66-NH2 composite nanoparticles by isothermal reaction; wherein, the dual-ligand peptide includes SEQ ID NO.1:GTTFSNYW and SEQ ID NO.2:MHYLEYPF, and the crosslinking agent includes o-phthalaldehyde (OPA); Step 2: The dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe was obtained by mixing and incubating the dual-ligand peptide / UiO-66-NH2 composite nanoparticles with the fluorescent signal molecule acridine yellow G (AYG) at room temperature.

2. The preparation method according to claim 1, characterized in that, Step 1 includes mixing UiO-66-NH2, GTTFSNYW, MHYLEYPF, and o-phthalaldehyde in proportion, ultrasonically dispersing for 1-3 min, purging with nitrogen for 4-6 min, and reacting under sealed conditions in a water bath at 60-70°C. The reaction solvent is one of methanol, n-butanol, dimethyl sulfoxide, and N,N-dimethylformamide, preferably dimethyl sulfoxide. The reaction time is 6-30 h, preferably 18-24 h.

3. The preparation method according to any one of claims 1-2, characterized in that, Step 2 includes: the concentration of the dual-ligand peptide / UiO-66-NH2 composite nanoparticles is 50-500 μg / mL, the concentration of acridine yellow G is 5-15 μg / mL, the incubation time at room temperature is 20-40 min, and the reaction solvent is methanol.

4. The preparation method according to any one of claims 1-3, characterized in that, The molar ratio of GTTFSNYW to MHYLEYPF in the dual-ligand peptide is 1:

1.

5. The preparation method according to any one of claims 1-4, characterized in that, The molar ratio of GTTFSNYW, MHYLEYPF, and o-phthalaldehyde is 1:1:(1-24); preferably 1:1:(12-24), and more preferably 1:1:

18.

6. The preparation method according to any one of claims 1-5, characterized in that, The molar ratio of UiO-66-NH2, GTTFSNYW, MHYLEYPF and o-phthalaldehyde is 1:1:1:(1-24), preferably 1:1:1:(12-24), and more preferably 1:1:1:

18.

7. The preparation method according to any one of claims 1-6, characterized in that, Step 1 includes: mixing UiO-66-NH2, GTTFSNYW, MHYLEYPF, and phthalaldehyde in proportion, ultrasonically dispersing for 1-3 min, purging with nitrogen for 4-6 min, and reacting under sealed conditions in a water bath at 60-70°C. The reaction solvent is dimethyl sulfoxide, the reaction time is 24 h, and the molar ratio of UiO-66-NH2, GTTFSNYW, MHYLEYPF, and phthalaldehyde is 1:1:1:

18. Step 2 includes: reacting the dual-ligand peptide / UiO-66-NH2 composite nanoparticles with the fluorescent signal molecule acridine yellow G (AYG) to obtain a dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe, wherein the concentration of the dual-ligand peptide / UiO-66-NH2 composite nanoparticles is 500 μg / mL, the concentration of acridine yellow G is 15 μg / mL, the mixture is mixed and incubated at room temperature for 20–40 min, and the reaction solvent is methanol.

8. A dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe prepared by the method according to any one of claims 1-7.

9. The application of a dual-ligand peptide / UiO-66-NH2@AYG fluorescent probe prepared by any one of claims 1-7 in the detection of CA153.

10. The application according to claim 9, characterized in that, The detection targets include spiked mouse serum samples or serum samples from clinical cancer patients. During detection, the fluorescent probe and the sample are incubated in the dark with shaking for 10–40 min, and the adsorption equilibrium time is 10–30 min.

Images