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Amplex red is the most widely used fluorescence substrate to HRP; however, it suffers from some drawbacks, such as nonspecific responsiveness toward carboxylesterases. Discovering a new small molecular fluorescence substrate with improved sensitivity and selectivity for HRP is thus desired. Herein, three dihydrofluorescein derivatives (DCFHs) are presented to serve as HRP substrates through fluorescence turn-on methods. The most promising one, 2,7-dichloro-9-(2-(hydroxymethyl)phenyl)-9H-xanthene-3,6-diol (DCFH-1), exhibited excellent sensitivity in the detection of HRP. Moreover, DCFH-1 does not respond to carboxylesterase, thus holding advantages over Amplex red. In the further study, the detection reagent in the commercial ELISA kits was replaced with DCFH-1 to establish a new fluorescence ELISA, which works very well in the quantification of inflammatory cytokine biomarkers from in vitro models.<\/jats:p>","DOI":"10.3390\/chemosensors11020152","type":"journal-article","created":{"date-parts":[[2023,2,21]],"date-time":"2023-02-21T01:39:28Z","timestamp":1676943568000},"page":"152","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Discovering a Dihydrofluorescein Analogue as a Promising Fluorescence Substrate to HRP"],"prefix":"10.3390","volume":"11","author":[{"given":"Jiayan","family":"Zhu","sequence":"first","affiliation":[{"name":"The First Clinical Medical College and the First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510006, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ting","family":"Li","sequence":"additional","affiliation":[{"name":"Artemisinin Research Center, Guangzhou University of Chinese Medicine, Guangzhou 510006, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shihui","family":"Zhang","sequence":"additional","affiliation":[{"name":"Artemisinin Research Center, Guangzhou University of Chinese Medicine, Guangzhou 510006, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaomei","family":"Zou","sequence":"additional","affiliation":[{"name":"The First Clinical Medical College and the First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510006, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yingchun","family":"Zhou","sequence":"additional","affiliation":[{"name":"The First Clinical Medical College and the First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510006, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Weiguo","family":"Lu","sequence":"additional","affiliation":[{"name":"The First Clinical Medical College and the First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510006, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhihui","family":"Liu","sequence":"additional","affiliation":[{"name":"The First Clinical Medical College and the First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510006, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tao","family":"Deng","sequence":"additional","affiliation":[{"name":"Artemisinin Research Center, Guangzhou University of Chinese Medicine, Guangzhou 510006, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1707-5183","authenticated-orcid":false,"given":"Fang","family":"Liu","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou 510006, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,2,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"6277","DOI":"10.1021\/ja9008858","article-title":"Mechanistic aspects of horseradish peroxidase elucidated through single-molecule studies","volume":"131","author":"Gorris","year":"2009","journal-title":"J. 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