{"title":"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。","authors":"Xueguo Liu, Minglin Lei, Yizhuo Zhao, Xueying Meng, Dongwan Li, Keyi Zhao, Shuqi Sun, Huanhuan Xing, Xiaojing Xing","doi":"10.1007/s00216-025-06088-4","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":462,"journal":{"name":"Analytical and Bioanalytical Chemistry","volume":" ","pages":""},"PeriodicalIF":3.8000,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Highly sensitive and high-throughput detection of human alkyladenine DNA glycosylase via hybridization chain reaction-assisted quantum dot fluorescent assay.\",\"authors\":\"Xueguo Liu, Minglin Lei, Yizhuo Zhao, Xueying Meng, Dongwan Li, Keyi Zhao, Shuqi Sun, Huanhuan Xing, Xiaojing Xing\",\"doi\":\"10.1007/s00216-025-06088-4\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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.</p>\",\"PeriodicalId\":462,\"journal\":{\"name\":\"Analytical and Bioanalytical Chemistry\",\"volume\":\" \",\"pages\":\"\"},\"PeriodicalIF\":3.8000,\"publicationDate\":\"2025-09-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Analytical and Bioanalytical Chemistry\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://doi.org/10.1007/s00216-025-06088-4\",\"RegionNum\":2,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Analytical and Bioanalytical Chemistry","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1007/s00216-025-06088-4","RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
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.
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.