A Peptide Fluorescent Probe Specifically Binding to Copper Ion and Cysteine

A fluorescent probe, species-specific technology, applied in the field of analysis and detection, can solve the problems of low probe sensitivity, poor biocompatibility, complicated preparation, etc., and achieve the effect of good biocompatibility, low toxicity, and simple preparation

Active Publication Date: 2020-05-15
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned probes generally have disadvantages such as low sensitivity, high toxicity, complicated preparation, poor biocompatibility, and high detection limit.

Method used

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  • A Peptide Fluorescent Probe Specifically Binding to Copper Ion and Cysteine
  • A Peptide Fluorescent Probe Specifically Binding to Copper Ion and Cysteine
  • A Peptide Fluorescent Probe Specifically Binding to Copper Ion and Cysteine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Polypeptide and polypeptide fluorescent probe P and polypeptide fluorescent probe P-Cu

[0035] 1.1 Preparation method

[0036] (1) Add 100 mg of Rink ammonia resin to a reactor with a sieve plate, use 5 mL of dichloromethane to swell the resin; remove N-terminal Fmoc from 20% piperidine / N,N-dimethylformamide (DMF) solution Protecting group, chromogenic reaction detects that the protecting group is completely removed;

[0037] (2) Dissolve 4eq N-terminal Fmoc-protected amino acids with 4eq HOBt and 4eq HBTU in DMF, activate at low temperature for 20min, add 6eq DIEA dropwise to the solution, mix the solution and add it to the reactor, and react for 3hrs;

[0038](3) After the reaction was finished, the reaction liquid was extracted from the reactor, and the resin was washed three times with 5 mL of DMF and DCM respectively. The complete condensation of amino acids was detected by color reaction, and the resin was treated with 20% piperidine / DMF solution for ...

Embodiment 2

[0046] Example 2 Polypeptide fluorescent probe P to Cu 2+ Specificity studies of detection

[0047] 2.1 Evaluation experiment of cation selectivity of polypeptide fluorescent probe P

[0048] In HEPES buffer (10mM, pH7.4), add 10 -5 mol / L polypeptide fluorescent probe P and 10 -5 mol / L metal cation (Ag + , Al 3+ , Ca 2+ , Cd 2+ ,Co 2+ , Cr 3+ , Cu 2+ , Fe 3+ , Hg 2+ , K + , Mg 2+ , Mn 2+ , Na + , Ni 2+ , Pb 2+ ), to detect the change in the fluorescence emission spectrum of the solution. The result is as figure 2 As shown, only Cu 2+ In the presence of , the fluorescence of the probe P was significantly quenched, and the other 15 metal ions could not quench the fluorescence of the polypeptide fluorescent probe P, indicating that the polypeptide should be able to recognize Cu 2+ .

[0049] 2.2 Polypeptide fluorescent probe P versus Cu2 + Specific detection research

[0050] In HEPES buffer (10mM, pH7.4), add 10 -5 mol / L polypeptide fluorescent probe P a...

Embodiment 3

[0057] Example 3 Specificity Research of Polypeptide Fluorescent Probe P-Cu for Cysteine ​​(Cys) Detection

[0058] 3.1 Selectivity evaluation experiment of polypeptide fluorescent probe P-Cu for amino acid detection

[0059] To HEPES buffer (10mM, pH7.4), add 10 -5 mol / L peptide fluorescent probe P-Cu and 10 - 5 mol / L cysteine ​​(Cys), glutamic acid (Glu), aspartic acid (Asp), asparagine (Asn), phenylalanine (Phe), alanine (Ala), gluten Aminoamide (Gln), Threonine (Thr), Serine (Ser), Tyrosine (Tyr), Lysine (Lys), Tryptophan (Trp), Proline (Pro), Glycine (Gly) , valine (Val), methionine (Met), leucine (Leu), isoleucine (Ile), arginine (Arg), histidine (His), glutathione (GSH) and S 2- , to detect changes in the fluorescence emission spectrum of the solution. The result is as Figure 6 As shown, only the addition of Cys causes the fluorescence intensity to become significant, while other substances do not cause significant changes in fluorescence intensity.

[0060] 3....

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Abstract

The invention discloses a polypeptide fluorescence probe specifically binding cupric ions with cysteine. The amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1; an N end of the polypeptide is modified with a fluorescent luminescent substance to form the polypeptide fluorescence probe P, the probe is capable of targeting and binding the Cu2+, has good selectivity, low toxicity and low detection limit, is simple to prepare and convenient to detect, and provides an efficient detection method for detecting cupric ions in biological and environmental samples; and the probe P is combined with the Cu2+ to form a P-Cu probe, and the probe can specifically identify Cys, and provide an efficient detection method for the rapid detection of the Cys in living bodies and environmental samples.

Description

technical field [0001] The invention belongs to the technical field of analysis and detection, and in particular relates to a polypeptide fluorescent probe specifically binding to copper ions and cysteine. Background technique [0002] Cu 2+ Cys (cysteine) is an indispensable small molecule in the human body and participates in the regulation of various physiological activities in the body. Cu 2+ It plays an important role in gene expression, immune regulation, and hematopoiesis, and an appropriate amount of Cu 2+ It will promote the physiological process of the body, but when Cu in the body 2+ When excessive, it will accumulate in the liver and cause liver cirrhosis, liver ascites, and diseases such as Alzheimer's disease and Down's syndrome are also related to the imbalance of copper metabolism in the body. Cys plays a vital role in biological functions such as protein synthesis and translation modification in organisms. When the Cys in the body is insufficient, it wi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64C09K11/06C07K7/06
CPCC07K7/06C09K11/06C09K2211/14G01N21/6428G01N21/643G01N21/6486G01N2021/6432
Inventor 肖建喜胡悦
Owner LANZHOU UNIVERSITY
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