杂交链反应辅助量子点荧光法检测人烷基腺嘌呤DNA糖基化酶的高灵敏度和高通量。

IF 3.8 2区 化学 Q1 BIOCHEMICAL RESEARCH METHODS
Xueguo Liu, Minglin Lei, Yizhuo Zhao, Xueying Meng, Dongwan Li, Keyi Zhao, Shuqi Sun, Huanhuan Xing, Xiaojing Xing
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引用次数: 0

摘要

人烷基腺嘌呤DNA糖基化酶(hAAG)是碱基切除修复(BER)通路中的重要酶,其异常表达与人类多种疾病有关。虽然已经开发了几种用于hAAG检测的方法,但构建低背景、高灵敏度和高通量的技术仍然是一个重大挑战。在此,我们引入了一个高灵敏度和高通量的hAAG活性检测平台,利用量子点(QDs)作为信号敏化剂,杂交链反应(HCR)进行信号放大,微孔板孔进行高通量分析。定制的带有糖基酶识别位点的发夹DNA底物在加入hAAG和APE1后发生构象变化,从而产生引物链。这些释放的引物链随后启动hcr介导的信号扩增,为dna功能化QD (DNA-QD)探针创造了许多结合位点。由于发夹衬底结构稳定,该方法显示出最小的背景信号,具有良好的灵敏度,检测限为0.012 U/mL。值得注意的是,该策略能够对hAAG抑制剂进行多种评估,并检测来自癌细胞的内源性hAAG,突出了其早期临床诊断的潜力。此外,该策略可以通过合理修饰DNA底物来量化各种DNA修复酶。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Highly sensitive and high-throughput detection of human alkyladenine DNA glycosylase via hybridization chain reaction-assisted quantum dot fluorescent assay.

Human alkyladenine DNA glycosylase (hAAG) is an important enzyme in the base excision repair (BER) pathway, and its abnormal expression is correlated with various human diseases. While several methods have been developed for hAAG detection, constructing low-background, highly sensitive, and high-throughput techniques remains a significant challenge. Herein, we introduce a highly sensitive and high-throughput platform for hAAG activity detection, utilizing quantum dots (QDs) as the signal sensitizer, the hybridization chain reaction (HCR) for signal amplification, and microplate wells for high-throughput analysis. The custom-designed hairpin DNA substrate with a glycosylase recognition site undergoes a conformational change upon the addition of hAAG and apurinic/apyrimidinic endonuclease 1 (APE1), resulting in the generation of primer chains. These released primer chains then initiate HCR-mediated signal amplification, creating numerous binding sites for DNA-functionalized QD (DNA-QD) probes. This method displays minimal background signal owing to the stable structure of the hairpin substrate and demonstrates excellent sensitivity with a detection limit of 0.012 U/mL. Notably, this strategy enables versatile evaluation of the hAAG inhibitor as well as the detection of endogenous hAAG from cancer cells, highlighting its potential for early clinical diagnosis. Additionally, this strategy could be adapted to quantify various DNA repair enzymes by rationally modifying the DNA substrates.

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来源期刊
CiteScore
8.00
自引率
4.70%
发文量
638
审稿时长
2.1 months
期刊介绍: Analytical and Bioanalytical Chemistry’s mission is the rapid publication of excellent and high-impact research articles on fundamental and applied topics of analytical and bioanalytical measurement science. Its scope is broad, and ranges from novel measurement platforms and their characterization to multidisciplinary approaches that effectively address important scientific problems. The Editors encourage submissions presenting innovative analytical research in concept, instrumentation, methods, and/or applications, including: mass spectrometry, spectroscopy, and electroanalysis; advanced separations; analytical strategies in “-omics” and imaging, bioanalysis, and sampling; miniaturized devices, medical diagnostics, sensors; analytical characterization of nano- and biomaterials; chemometrics and advanced data analysis.
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