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Microfluidic chip for instantly detecting EGFR (epidermal growth factor receptor) mutation

A microfluidic chip and substrate technology, applied in the field of clinical medicine, can solve the problem that the micro-LAMP system is not fully compatible

Active Publication Date: 2015-05-06
THE SECOND HOSPITAL OF DALIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the micro-LAMP system does not fully meet the standards proposed by the WHO in terms of no need for large-scale instruments (no need for large-scale instruments such as electrophoresis instruments) and operation.

Method used

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  • Microfluidic chip for instantly detecting EGFR (epidermal growth factor receptor) mutation
  • Microfluidic chip for instantly detecting EGFR (epidermal growth factor receptor) mutation
  • Microfluidic chip for instantly detecting EGFR (epidermal growth factor receptor) mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 A microfluidic chip

[0073] The microfluidic chip is an airtight whole formed by bonding the upper substrate 1 and the lower substrate 2; the microfluidic chip is provided with three identical and independent LAMP systems; each LAMP system includes an airbag chamber 11, A small chamber 21 equipped with constant temperature amplification buffer, a small chamber 22 equipped with primers, a small chamber 23 equipped with Bst DNA polymerase, a LAMP reaction chamber 15, a gas channel 16, a gas channel 17, a gas channel 18; the upper half of the airbag chamber 11, the upper half of the chamber 21 equipped with constant temperature amplification buffer, the upper half of the chamber 22 equipped with primers, the upper half of the chamber 23 equipped with Bst DNA polymerase, The upper part of the LAMP reaction chamber 15, the gas channel 16, the gas channel 17, and the gas channel 18 are located on the upper substrate 1, and one end of the gas channel 16 communicates...

Embodiment 2

[0096] Example 2 The preparation method of the microfluidic chip in Example 1

[0097] step:

[0098] 1. Prepare the SU-8 positive mold with microchannels and microstructures on the microfluidic chip in Example 1

[0099] Firstly, the microchannel and microstructure of the microfluidic chip were designed with computer-aided design software (Freehand); secondly, the microchannel map drawn by Freehand was printed on SU-8 glue (Microchem, model 2075) as a mask, and the The standard photolithography process is used to make the mold, and the SU-8 positive mold is obtained.

[0100] 2. PDMS replication

[0101] (1) Mix PDMS (Dow Corning, article number: 0007883528) and curing agent at a mass ratio of 10:1, pump air in a vacuum drying oven, pour it on the surface of the mold, bake at 80°C for 1 hour, and take it out;

[0102] (2) Slowly tear off the PDMS on the template after cooling, drill the injection hole and waste liquid hole at the corresponding position of the PDMS substrat...

Embodiment 3

[0107] Example 3 Research on the effect of the microfluidic chip in Example 1 in detecting EGFR gene point mutations

[0108] step:

[0109] 1. Cell collection and lysis

[0110] Take a bottle of H1975 with a confluence of 80%-90% (there is a point mutation in exon 21 of the EGFP gene on the cell genome) and a bottle of the negative control human lung adenocarcinoma cell line A549. After washing with PBS, add 1.5 mL of DNAiso reagent (Takara), shake the cell culture flask gently.

[0111] 2. Detection of point mutations in exon 21 of EGFP gene on sample DNA

[0112] (1) Use a syringe to suck out the lysate containing cell fragments, inject the above-mentioned cell lysate into the LAMP reaction chamber 15 through the sample inlet 181 in the sample LAMP system on the microfluidic chip, and let it stand for several minutes to make the nucleic acid released by cell lysis Adsorbed to the surface of the glass frit, then inject air into the sample inlet 181 with a syringe, so that...

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Abstract

The invention provides a microfluidic chip for instantly detecting EGFR (epidermal growth factor receptor) mutation. The microfluidic chip integrates the functions of nucleic acid extraction, LAMP (loop-mediated isothermal amplification) and instant detection. The microfluidic chip solves the problem of centrifugation step required by DNA (deoxyribonucleic acid) extraction before constant-temperature DNA amplification and the problem of thermal circulation apparatus required by DNA amplification, and conforms to the general requirements for diagnostic detection in underdeveloped regions provided by WHO (World Health Organization).

Description

technical field [0001] The invention belongs to the field of clinical medicine application and relates to a microfluidic chip for real-time detection of EGFR mutation. The microfluidic chip of the present invention integrates nucleic acid extraction, LAMP amplification, and real-time detection. Background technique [0002] Lung cancer has become the malignant tumor with the highest morbidity and mortality at home and abroad. A large number of studies have shown that epidermal growth factor receptor (EGFR), as a member of the receptor tyrosine superfamily, is expressed in a variety of malignant tumors. The combination of ligand and EGFR induces the formation of dimers, changes the conformation, activates tyrosine kinases and signal transduction pathways, and produces effects such as cell proliferation, invasion, metastasis and anti-apoptosis. Clinical studies have shown that activating mutations in the kinase domain of EGFR are associated with drug sensitivity, making only...

Claims

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Application Information

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IPC IPC(8): C12M1/34C12Q1/68
Inventor 王琪吕建新杜小慧赵阳刘元斌郭哲
Owner THE SECOND HOSPITAL OF DALIAN MEDICAL UNIV
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