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Detection method of electrochemical acute leukemia gene Pax-5a based on enzyme assisted cyclic signal amplification

A technology of acute leukemia and pax-5a, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as limiting detection sensitivity, and achieve the effects of avoiding detection interference, improving reaction speed, and reducing complexity

Active Publication Date: 2019-11-05
JIANGXI NORMAL UNIV
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Problems solved by technology

[0006]However, electrochemical biosensors generally have certain limitations in terms of signal generation
On the one hand, when constructing electrochemical biosensors, appropriate electroactive substances need to be labeled, which may limit the detection sensitivity; on the other hand, general electrochemical layer-by-layer assembly reactions will continuously introduce interference substances on the electrode surface; thirdly, Incorporating appropriate signal amplification techniques into the construction of electrochemical biosensors directly at the electrode interface is an efficiency issue

Method used

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  • Detection method of electrochemical acute leukemia gene Pax-5a based on enzyme assisted cyclic signal amplification
  • Detection method of electrochemical acute leukemia gene Pax-5a based on enzyme assisted cyclic signal amplification
  • Detection method of electrochemical acute leukemia gene Pax-5a based on enzyme assisted cyclic signal amplification

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Embodiment 1

[0035] (1) Treat the thiol-modified probe (TP) with 2mM TCEP at 37°C for 2 hours to reduce the presence of disulfide bonds; then, add 10 μL of 0.3 μM treated thiol-modified probe (TP) solution dropwise To the surface of the gold electrode, incubate at 4°C for 12 hours, then rinse the electrode with Tris-HCl buffer (10mM, 1mM EDTA) to remove unbound TP probes, and block the electrode with 2mM MCH to obtain the treated modified Gold electrode MCH / TP / AuE;

[0036](2) Put 2 μL of hairpin probe (HP) with a concentration of 10 μM and 1 μL of Pax-5a standard aqueous solution with a concentration of 500pM, 100pM, 50pM, 10pM, 5pM, 1pM, 500fM, 100fM, 10fM, and 0fM in the reaction tube Mix in medium, add CutSmart buffer (20mM Tris-HAc, 50mM KAc, 10mM MgAc2, 0.1g / mL BSA, pH 7.9), incubate at 37°C for 1 hour; Add 5U / μL of Klenow Fragment, 1μL of 10mM dNTPs and 1μL of 10U / μL Nt.BbvCI, incubate at 37°C for 3 hours, then heat at 80°C for 20 minutes to inactivate the enzyme;

[0037] (3) Tra...

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Abstract

Provided is a detection method of an electrochemical acute leukemia gene Pax-5a based on enzyme assisted cyclic signal amplification; the efficiency advantage of transcription termination and translation is controlled based on a hairpin DNA probe (HP). The target gene Pax-5a opens HP, and the replication template function of the HP is started. Under the synergistic effect of DNA polymerase and restriction endonuclease, on one hand, auxiliary DNA promotes the target gene Pax-5a to cyclically open the HP; on the other end, a large number of G-quadruplex sequences are produced. The G-quadruplex sequences are captured by a sulfhydryl-modified probe on the surface of a gold electrode due to the complementary action of bases, then a G-quadruplex / hemin complex is formed in the presence of hematinchloride and potassium ions, and hydrogen peroxide can be catalyzed to reduce so as to generate electrochemical signals; the change of the current has linear correlation relationship with the concentration of the target gene in a certain range. Based on the mechanism of enzyme assisted cyclic signal amplification of the hairpin DNA probe and a one-key electrochemical detection platform, high-sensitivity and high-selectivity detection of the target gene can be achieved.

Description

technical field [0001] The invention relates to the field of electrochemical biological analysis, in particular to a detection method for acute leukemia gene Pax-5a. Background technique [0002] As one of the most common human malignancies, acute lymphoblastic leukemia (ALL) has the highest prevalence among children. About 3,000 to 4,000 people are diagnosed with ALL each year in the United States, two-thirds of whom are children between the ages of 2 and 5. Pax-5 is the only paired-box (PAX) family member found in the hematopoietic system, which can be divided into Pax-5a, Pax-5b, Pax-5c, Pax-5d and Pax-5e. Among them, Pax-5a is the most important, and Pax-5 usually refers to Pax-5a. Pax-5 is very important for the differentiation and development of B cells, and its abnormal expression can cause B lymphocytic leukemia. However, there are few studies on the detection of variant Pax5 gene and the expression of this gene in clinical acute lymphoblastic leukemia patients. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/682C12Q1/6825
CPCC12Q1/682C12Q1/6825C12Q2521/101C12Q2521/301C12Q2565/607
Inventor 黎泓波刘明彬赵卫华王素琴
Owner JIANGXI NORMAL UNIV
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