Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Caspase biosensor, application thereof and detection method of caspase activity

A caspase and biosensor technology, applied in the field of bioanalysis, can solve problems such as false positive signals, low detection sensitivity of caspase, increased measurement cost and complexity of probe synthesis

Active Publication Date: 2020-06-19
SHANDONG NORMAL UNIV
View PDF3 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the inventors of the present invention have found that these fluorescent detection methods often use fluorescent labels (including fluorescent proteins and organic dyes, etc.) and quenched labeled peptides to detect the activity of caspases as enzyme substrates, not only Increases the cost of the assay and the complexity of probe synthesis, and may also lead to false positive signals due to incomplete quenching of fluorescence; at the same time, the detection sensitivity of current fluorescence methods for caspase activity is still low

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Caspase biosensor, application thereof and detection method of caspase activity
  • Caspase biosensor, application thereof and detection method of caspase activity
  • Caspase biosensor, application thereof and detection method of caspase activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0044] The third embodiment of the present invention provides a kit for detecting caspase activity, comprising the above-mentioned caspase biosensor, buffer, and deoxyribonucleoside triphosphate.

[0045] The fourth embodiment of the present invention provides a method for detecting caspase activity, providing the above-mentioned caspase biosensor or kit, which enables the detection of caspase activity through the interaction of streptavidin and biotin The probe is connected to the magnetic beads, the caspase recognizes the specific site of the detection probe and cuts it, releasing the DNA primer domain, and the released DNA primer domain performs branch rolling circle with the circular template, DNA polymerase, and secondary primer Amplification reaction followed by addition of fluorochrome for fluorescence detection.

[0046] The method for detecting caspase activity of the present invention is preferably aimed at the diagnosis and treatment of non-diseases.

[0047] In on...

Embodiment 1

[0058] Preparation and testing of materials:

[0059] Cell culture and preparation of cell extracts: Human cervical cancer cells (HeLa) and human breast cancer cells (MCF-7) were modified with Dalbec containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody, respectively. Eagle's medium (DMEM) was cultured at 37°C in an incubator containing 5% carbon dioxide. For actual sample analysis, cells in the exponential growth phase were collected by trypsinization, and live cell counts were performed using an automatic cell counter IC1000 from Countstar Biotechnology Co., Ltd., washed twice with ice-cold 1X working concentration of PBS and then rotated at 800 Centrifuge for 5 minutes at a speed of 1 minute. Cells were then resuspended in 100 microliters of lysis buffer (containing 150 millimolar NaCl, 1% NP-40, 0.25 millimole sodium deoxycholate, 1.0 percent glycerol, 0.1 millimolar per liter of 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride and 10 mm...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Excitation wavelengthaaaaaaaaaa
Login to View More

Abstract

The invention discloses a caspase biosensor, application thereof and a detection method of caspase activity. The caspase biosensor comprises a magnetic bead, a detection probe, an annular template, DNA polymerase and a secondary primer, wherein the surface of the magnetic bead is coated with streptavidin; the detection probe comprises a peptide substrate structural domain and a DNA primer structural domain which are connected, the peptide substrate structural domain is an amino acid sequence, and the DNA primer structural domain is a single-stranded DNA sequence; the peptide substrate structural domain contains a specific site, the specific site can be recognized and cut by caspase, the specific site and a connecting site are separated by n amino acids, the connecting site is a joint of the peptide substrate structural domain and the DNA primer structural domain, the peptide substrate structural domain is connected with biotin, the specific site is positioned between the biotin and theconnecting site, and n is a natural number smaller than 10; and the DNA primer structural domain can be matched with the annular template, the DNA polymerase and the secondary primer for a branch rolling circle amplification reaction.

Description

technical field [0001] The invention belongs to the technical field of bioanalysis, and relates to a caspase biosensor and its application, and a method for detecting caspase activity. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Apoptosis is an important programmed cell death process that regulates a series of important physiological processes and stress responses by eliminating unwanted cells in the body. Cysteine-aspartyl-specific proteases (caspases) are considered to be the main executors of apoptosis by efficiently cleaving specific cellular substrates such as poly ADP-ribose polymerase and nuclear fibrinolytic enzymes) lead to apoptosis. Abnormal activity of c...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/6844C12Q1/37
CPCC12Q1/6844C12Q1/37C12Q2531/125C12Q2565/607C12Q2563/143C12Q2563/149C12Q2563/107
Inventor 张春阳刘萌张迪马飞
Owner SHANDONG NORMAL UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com