Label-free Fluorescence Probe Based on Primer Exchange Reaction for High Sensitivity Detection of Apurinic/Apyrimidinic Endonuclease 1
10.19756/j.issn.0253-3820.241457
- VernacularTitle:基于引物交换反应的非标记荧光探针用于脱嘌呤/脱嘧啶核酸内切酶1的高灵敏检测
- Author:
Yun-Hua WANG
1
;
Le-Ru WANG
;
Li-Gai YANG
;
Jia-Zheng CHEN
;
Yu-Run DU
;
Jia-Hui HOU
;
Xiang ZHAI
;
Xu-Hua ZHAO
;
Bao-Feng YU
Author Information
1. 山西医科大学基础医学院,生物化学与分子生物学教研室,太原 030001
- Keywords:
Apurinic/apyrimidinic endonuclease 1;
Primer exchange reaction;
G-quadruplex;
Thioflavin T;
Label-free fluorescence probe
- From:
Chinese Journal of Analytical Chemistry
2025;53(3):464-471
- CountryChina
- Language:Chinese
-
Abstract:
Apurinic/apyrimidinic endonuclease 1(APE 1)is a multifunctional protein that plays important roles in DNA repair and regulation of gene expression.Because APE 1 is overexpressed in various cancers,it can serve as a cancer biomarker for aiding clinical diagnosis,guiding therapy,and monitoring prognosis.On this basis,a label-free fluorescent probe was designed based on the primer exchange reaction(PER)strategy for highly sensitive detection of APE 1 activity.In the absence of APE 1,the structure of catalytic hairpin(HP)was stable and could not form G-quadruplex.Therefore,the background fluorescence of this sensing system was very low due to the dissociation of thioflavin T(ThT).In the presence of APE 1,the apurinic/apyrimidinic(AP)site of HP was cleaved by APE 1 and a short nucleic acid fragment that acted as a primer to initiate PER was generated.After PER reaction,a large number of G-quadruplex were produced,which could specifically bind with ThT and resulted in significant increase of fluorescence signal.The combination of low background design of HP and PER amplification made this biosensor had high sensitivity with a detection limit(3σ)of 0.0008 U/mL.Furthermore,the primer sequence was directly generated by the cleavage of APE 1 without additional addition,which not only increased the specificity of the reaction,but also simplified the experiment procedure.Moreover,the use of label-free fluorescence signal reduced the cost of the experiment,and realized rapid detection of APE 1.Finally,this sensor was used to detect APE 1 in human serum samples with spiked recoveries of 91%-104%,proving great potential in study of biological enzyme.
