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A fluorescent probe for trypsin detection and its preparation method

A technology of fluorescent probe and trypsin, which is applied in the interdisciplinary field of biology and analytical chemistry, and in the field of materials, can solve unseen problems and achieve good stability, strong practicability, and simple and sensitive detection process

Inactive Publication Date: 2017-05-10
TAIYUAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Through searching, no pyrene-modified polymer fluorescence detection method for trypsin

Method used

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  • A fluorescent probe for trypsin detection and its preparation method
  • A fluorescent probe for trypsin detection and its preparation method
  • A fluorescent probe for trypsin detection and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach 1

[0023] (1) Preparation of fluorescent probes

[0024] ① The fluorescently labeled polymer is pyrene-terminated polyethylene oxide with the following chemical structure. Dissolving the fluorescently labeled polymer in a phosphate buffer solution with a concentration of 10 mM and pH=7.4 to prepare an aqueous solution A with a concentration of 0.0002% by weight;

[0025]

[0026] ② Dissolve cytochrome C derived from pig heart in phosphate buffer solution with a concentration of 10mM and pH=7.4 to prepare an aqueous solution B with a concentration of 0.0008% by weight.

[0027] ③ Mix 2 mL of aqueous solution A with 2 mL of aqueous solution B and react for 5 minutes to allow cytochrome C to adsorb on the surface of the fluorescently labeled polymer to obtain a fluorescent probe for detecting trypsin.

[0028] The fluorescence spectrum showed that the addition of cytochrome C would reduce the fluorescence intensity of the fluorescently labeled polymer, indicating that cytochrome...

Embodiment approach 2

[0036] (1) Preparation of fluorescent probes

[0037] Except that the fluorescent labeling polymer used is the pyrene-terminated poly-2-methacryloyloxyethyl phosphorylcholine with the following chemical structural formula, and the mixed reaction of aqueous solution A and aqueous solution B is 10 minutes, other is the same as embodiment 1.

[0038]

[0039] The fluorescence spectrum showed that the addition of cytochrome C would reduce the fluorescence intensity of the fluorescently labeled polymer, indicating that cytochrome C was adsorbed to the surface of the fluorescently labeled polymer.

[0040] (2) Detection of trypsin: with embodiment 1.

[0041] The detection results of fluorescence spectrum showed that the decrease ratio of fluorescence intensity at the emission peak position had a good linear relationship with the concentration of trypsin, and the fluorescent probe could detect trypsin in the buffer solution. The detection has good specificity, and other proteins...

Embodiment approach 3

[0043] (1) Preparation of fluorescent probes

[0044] ① The fluorescently labeled polymer is pyrene-terminated polyethylene oxide with the following chemical structure. Dissolving the fluorescently labeled polymer in a Tris-HCl buffer solution with a concentration of 50 mM and pH=8.0 to prepare an aqueous solution A with a concentration of 0.005% by weight;

[0045]

[0046] ② dissolving cytochrome C derived from horse heart in Tris-HCl buffer solution with a concentration of 50 mM and pH=8.0 to prepare an aqueous solution B with a concentration of 0.025% by weight;

[0047] ③ except that the mixing reaction time is 15 minutes, other is the same as embodiment 1.

[0048] The fluorescence spectrum showed that the addition of cytochrome C would reduce the fluorescence intensity of the fluorescently labeled polymer, indicating that cytochrome C was adsorbed to the surface of the fluorescently labeled polymer.

[0049] (2) Detection of trypsin: except the used buffer solution...

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Abstract

The invention relates to a fluorescent probe for trypsin detection and a preparing method of the fluorescent probe, and belongs to the technical field of material, biology and analytic chemistry interdiscipline. The fluorescent probe is characterized in that the fluorescent probe is a composition water solution composed of fluorescent mark polymer and cytochrome C adsorbed to the surface of the fluorescent mark polymer, the weight percentage concentration of the composition water solution is 0.0005%-0.05%, hydrophilic polymer modification is carried out on hydrophobicity dye pyrene, the obtained water solubility fluorescent mark polymer serves as the fluorescence-emission energy donor, the cytochrome C serves as the substrate of the trypsin, and the fluorescent probe for trypsin detection is obtained through the adsorption of the cytochrome C on the surface of the fluorescent mark polymer. The trypsin with the hydrolysis on the cytochrome C is added, the heme obtained through hydrolysis serves as the energy receptor of fluorescence, and the detection of the enzymatic activity is achieved through reduction of the intensity of the fluorescence. The fluorescent probe has high fluorescence strength and good water solubility and stability and has wide application prospects in the field of biosensors.

Description

technical field [0001] The invention discloses a fluorescent probe for detecting trypsin and a preparation method thereof, belonging to the technical field of interdisciplinary materials, biology and analytical chemistry. Background technique [0002] Trypsin is one of the most important digestive enzymes in protein decomposition. The secretion, activation, inhibition and circulation imbalance of trypsinogen can lead to acute or chronic pancreatic diseases, such as pancreatic cancer. Approximately 35,000 cases of pancreatic cancer each year are caused by an imbalance of serine proteases, especially trypsin. In addition, trypsin not only acts as a digestive enzyme, but also restricts the decomposition of precursors of other enzymes such as chymotrypsinogen, carboxypeptidase, and phospholipase, and activates them. [0003] Currently developed methods for the determination of trypsin mainly include UV spectrophotometry, colorimetry, fluorescence, immunoassay, mass spectrometry...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C09K11/06G01N21/64
Inventor 贾兰陈松刘祥侯文娟张耀东
Owner TAIYUAN UNIV OF TECH