Preparation method and application of polypeptide fluorescent probe for detecting Cd < 2 + >
By designing a polypeptide fluorescent probe based on AIE aggregation-induced effect, the problem of difficulty in efficiently and selectively detecting Cd2+ in organisms in the prior art is solved, and qualitative detection under biological conditions is achieved, with low toxicity and high biocompatibility, and is suitable for the monitoring and diagnosis of cadmium-related diseases.
Patent Information
- Application Number
- CN202411440642.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-16
- Publication Date
- 2025-03-18
AI Technical Summary
The prior art is difficult to efficiently and selectively detect the aggregation of Cd2+ in organisms, especially in complex biological systems.
A fluorescent probe of polypeptide based on AIE aggregation-induced effect was designed to achieve high selective recognition and detection of Cd2+ through a specific coordination and binding mechanism. The probe consists of specific amino acid sequences, which combine with tetraphenylethylene (TPE) derivatives, enhances the fluorescence response.
Qualitative detection of Cd2+ under biological conditions is achieved, with low toxicity and high biocompatibility, suitable for monitoring the aggregation of Cd2+ in plants and organisms, and has the potential to be used for pathological research and diagnosis of cadmium-related diseases.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biochemical medicine, and in particular to a method for detecting Cd 2+ The invention discloses a preparation method and application of a polypeptide fluorescent probe. Background Art
[0002] In the past two decades, the innovation of luminescent materials has opened up new avenues for science and technology, and aggregation-induced emission (AIE) is one of the important ways. Non-luminescent materials in the state of separated molecules can induce strong fluorescence when they aggregate, which has the opposite effect to the luminescence quenching (ACQ) effect caused by molecular aggregation. According to the hard-hard acid-base principle that hard acids tend to bind to hard bases and soft acids tend to bind to soft bases, histidine (His) easily binds to Zn 2+ ,Cd 2+ ,Cu 2+ ,Ni 2+ ,Ag + Coordination occurs, and cysteine (Cys) is more likely to bind to Hg 2+ ,Pb 2+ ,Zn 2+ ,Cd 2+ Coordination occurs, tyrosine (Try) and Cu 2+ It is easier to coordinate and design the polypeptide sequence. Compared with other polypeptide probes, the polypeptide probe composed of amino acids has the advantages of excellent performance and simple synthesis. At the same time, the addition of polypeptide sequences increases solubility and biocompatibility as well as low biological toxicity. Therefore, the present invention discloses a method for detecting Cd 2+ Preparation method of polypeptide fluorescent probe and its application for monitoring Cd in plants and organisms 2+ Aggregation, even for Cd 2+ Induced renal injury also has a monitoring role. Summary of the invention
[0003] Based on the current status of the above-mentioned prior art research, the present invention provides a method for detecting Cd 2+ The preparation method and use of the polypeptide fluorescent probe. The fluorescent probe of the present invention is for Cd 2+ Specific recognition can be used to monitor Cd in organisms 2+ of gathering.
[0004] Specifically, the present invention provides a fluorescent probe, the structure of which is shown in the following formula (I):
[0005]
[0006] The polypeptide fluorescent probe based on AIE aggregation induction effect of the present invention has the characteristics of high selectivity, low toxicity and good biocompatibility, so it is suitable for imaging under biological conditions and can be used to monitor Cd in plants and organisms. 2+ Aggregation, even for Cd 2+ It also has a monitoring effect on the renal damage induced by Cd. 2+ It can be used for qualitative detection of cadmium and is expected to be applied in the pathological research and diagnosis of other cadmium-related diseases.
[0007] The specific preparation method of the polypeptide fluorescent probe of the present invention is as follows: add dichlororesin to a polypeptide synthesis tube, add dichloromethane (DCM) thereto to swell the resin, and wash it with dimethylformamide (DMF). Add the corresponding amino acid mass (3.0eq.) and diethylenetriamine (DIEA) to react in a DMF solution for 2h. After the reaction is completed, wash with DMF and then use K's test (pyridine and ninhydrin reagent). Subsequently, the resin after the condensation reaction is deprotected from Fmoc for 15min, and washed with DMF solution and methanol solution respectively for K's test. The subsequent amino acid coupling is repeated according to the above steps, and the last amino acid is deprotected from Fmoc and then a tetraphenylethylene (TPE) derivative is added to react for 40min, and then washed and dried with DMF solution and methanol solution respectively. After the synthesis is completed, the polypeptide probe is cleaved from the resin with 87% TFA solution. First, treat with TFA solution at room temperature for 2h, filter, add ether for precipitation, centrifuge at 4000rpm, and dry to obtain a crude product. BRIEF DESCRIPTION OF THE DRAWINGS
[0008] Figure 1 Example 1 is for Cd 2+ Schematic diagram of the fluorescence spectrum of the sensing characteristics.
[0009] Figure 2 It is a schematic diagram of the cytotoxicity results of Example 1 on 293T cells (A) and HeLa cells (B).
[0010] Figure 3 Example 1 is the treatment of different concentrations of Cd in cells 2+ Schematic diagram of cell imaging (A) and relative fluorescence intensity (B). Experimental Example 1: Example 1 for Cd 2+ Selective fluorescence spectroscopy experiments.
[0011] Experimental method: Example 1 was dissolved in dimethyl sulfoxide (DMSO) to a 1.0 mM stock solution and stored at -20°C for the study of fluorescence spectroscopic properties. Ultrapure water was used to prepare 16 metal salts into 1.0 mM metal ion compounds (Cu 2+ ,Hg 2+ Mg 2+ , Pb 2+ ,Ni 2+ ,Al 3+ ,Ag + ,Na + ,K + ,Co 2+ ,Zn 2+ ,Ca 2+ ,Fe 3+ ,Ba 2+ ,Cr 3+ ,Cd 2+ ), and stored at low temperature for future use. In a buffer system (PBS, pH=7.4), 2 μL of Example 1 and metal ions were respectively mixed, and the mixture was made up to 200 μL with PBS buffer solution, and prepared at 37° C. for the determination of fluorescence spectral properties.
[0012] Experimental results: Example 1 for Cd 2+ The fluorescence response intensity of Cu is enhanced by more than four times compared with other ions. 2+ , Hg 2+ and Mg 2+ There is also fluorescence response, but they are different from Cd 2+ Compared with the previous study, the decrease was four times, indicating that Example 1 has a significant effect on Cd 2+ Specific selectivity.
[0013] Test Example 2: Test of cytotoxic activity of 293T cells and HeLa cells in Example 1
[0014] Experimental method: 1×10 4 293T cells and HeLa cells were treated with the compounds in Example 1 for 24 hours, and the cytotoxicity was determined by MTT method.
[0015] Experimental results: The results are as follows Figure 2 As shown, the results show that after the cells are treated with Example 1 at different concentrations, the survival rate of the cells reaches more than 90%, and the results confirm that the application of Example 1 in cells has low biotoxicity and high biocompatibility.
[0016] Test Example 3: Example 1 for Cd 2+ Cell imaging assay
[0017] Experimental method: 293T cells were seeded into a 12-well plate and cultured in a constant temperature incubator at 37°C and 5% CO2 for 48 h. Then, 10 μM of Example 1 was added and incubated for 1 h. The original culture medium was aspirated and washed once with PBS buffer solution. Then, 10 μM, 20 μM and 50 μM of Cd were added respectively. 2+After incubation for 30 min, the original culture medium was aspirated, and the cells were washed once with PBS buffer solution before cell imaging.
[0018] Experimental results: The results are as follows Figure 3 As shown, Example 1 and Cd 2+ After incubation with Cd 2+ The concentration of Cd was enhanced. This shows that Example 1 can track Cd in living cells. 2+ , which can be used as a highly sensitive peptide probe to detect Cd in solution and living cells 2+ potential.
Claims
1. The present invention discloses a method for detecting Cd 2+ Preparation method and use of polypeptide fluorescent probe, the detection of Cd 2+ The polypeptide fluorescent probe has a structure as shown in the following formula (I). The fluorescent probe of the present invention can be used for Cd 2+ Specific identification and monitoring of Cd in organisms 2+ Gathering of: （Ⅰ）。 2. A tetraphenylethylene (TPE) derivative, characterized in that: Its structure is shown in the following formula (I): （Ⅰ）。 3. A new use of the fluorescent probe as claimed in claim 1, characterized in that: Can be used for Cd 2+ Specific identification and monitoring of Cd in organisms 2+ of gathering.