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Method for detecting trypsin using unmarked fluorescence

A trypsin and fluorescence detection technology, which is applied in the interdisciplinary fields of analytical chemistry, biology, and materials, can solve unseen problems and achieve the effects of simple preparation, short detection time, and low cost

Inactive Publication Date: 2014-02-26
TAIYUAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Through searching, there is no method for label-free fluorescent detection of trypsin

Method used

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  • Method for detecting trypsin using unmarked fluorescence
  • Method for detecting trypsin using unmarked fluorescence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach 1

[0029] Ⅰ Preparation of organic solution of fluorescent probe:

[0030] Using pyrene as a fluorescent probe, dissolve pyrene in acetone and prepare a concentration of 10 in a 10 mL volumetric flask. -2 mol L -1 Pyrene in acetone, further diluted with acetone to 10 -5 mol L -1 ;

[0031] Ⅱ Determination of critical micelle concentration CMC of surfactant:

[0032] Dissolve sodium dodecyl sulfate SDS in a phosphate buffer solution with a concentration of 10 mM and pH 8.0, and prepare 10 in a 10 mL volumetric flask -2 mol L -1 The solution is further diluted with buffer solution to obtain a concentration range of 1mol L -1 ~10 -7 mol L -1 The gradient solution;

[0033] Use a microsampler to pipette the organic solution of the fluorescent probe prepared in step I, and add it to the prepared gradient solution respectively, so that the final concentration of the fluorescent probe in the gradient solution is 5×10 -7 mol L -1 . Ultrasonic treatment was performed at 100...

Embodiment approach 2

[0048] Ⅰ Preparation of organic solution of fluorescent probe:

[0049] Using Nile Red as a fluorescent probe, dissolve Nile Red in methanol and prepare a concentration of 10 in a 10 mL volumetric flask. -1 mol L -1 Methanol solution of Nile red, further diluted with methanol to 10 -3 mol L -1 ;

[0050] Ⅱ Determination of critical micelle concentration CMC of surfactant:

[0051] Dissolve sodium tetradecyl sulfate in a phosphate buffer solution with a concentration of 10 mM and pH 8.0, and prepare 10 in a 10 mL volumetric flask -2 mol L -1 The solution is further diluted with buffer solution to obtain a concentration range of 1mol L -1 ~10 -7 mol L -1 The gradient solution;

[0052] Use a microsampler to pipette the organic solution of the fluorescent probe prepared in step I, and add it to the prepared gradient solution respectively, so that the final concentration of the fluorescent probe in the gradient solution is 10 -6 mol L -1 . Ultrasonic treatment was...

Embodiment approach 3

[0067] Ⅰ Preparation of organic solution of fluorescent probe:

[0068] Using Sudan Red as a fluorescent probe, dissolve Sudan Red in chloroform and prepare a concentration of 10 in a 10 mL volumetric flask. -1 mol L -1 Chloroform solution of Sudan Red, further diluted with chloroform to 10 -3 mol L -1 ;

[0069] Ⅱ Determination of critical micelle concentration CMC of surfactant:

[0070] Sodium cetyl sulfate was dissolved in a phosphate buffer solution with a concentration of 10 mM and pH 8.0, and 10 -2 mol L -1 The solution is further diluted with buffer solution to obtain a concentration range of 1mol L -1 ~10 -7 mol L -1 The gradient solution;

[0071] Use a microsampler to pipette the organic solution of the fluorescent probe prepared in step I, and add it to the prepared gradient solution respectively, so that the final concentration of the fluorescent probe in the gradient solution is 10 -6 mol L -1 . Ultrasonic treatment was performed at 40 kHz for 60 ...

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Abstract

A method for detecting trypsin using unmarked fluorescence belongs to the technical field of interdisciplinary subjects of material, biology, and analytical chemistry, and particularly relates to the method for detecting the trypsin based on disassembly of supramolecular assemblies under enzymatic hydrolysis. The method is characterized by including: firstly, determining critical micellar concentration of a surfactant; secondly, forming the supramolecular assemblies with the surfactant and polyelectrolyte with opposite charges through hydrophobic and electrostatic interaction, and embedding hydrophobic dyes serving as fluorescent probes into the inner cavities of the supramolecular assemblies; thirdly, adding enzyme which can lead to hydrolysis in the polyelectrolyte so as to lead the disassembly of the supramolecular assemblies and release the embedded hydrophobic dyes, and detecting enzyme activity by lowering fluorescence intensity. The method is simple to prepare, low in cost, real-time in detection, and wide in application prospect in fields such as diagnosis and detection of biomolecules, and biosensors.

Description

technical field [0001] The invention discloses a method for detecting trypsin with unlabeled fluorescence, which belongs to the technical field of interdisciplinary materials, biology, and analytical chemistry, and specifically relates to a method for realizing trypsin detection based on supramolecular assembly disassembly under enzyme-catalyzed hydrolysis The technical solution of the method. 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. Pancreatic cancer is the most malignant tumor with the worst prognosis. The 5-year survival rate of patients is generally less than 5%. About 35,000 cases of pancreatic cancer are caused by the imbalance of serine proteases, especially trypsin, every year. In addition, trypsin not only acts as a digestive enzyme, but also ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64G01N21/75
Inventor 贾兰李文凤朱晶心刘晓华
Owner TAIYUAN UNIV OF TECH
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