Gong Tingting, Yan Huaifeng, Li Daxiu, Jiang Bingying, Xiang Yun, Yuan Ruo
School of Chemistry and Chemical Engineering, Chongqing University of Technology, Chongqing, 400054, PR China.
College of Pharmacy and Biological Engineering, Chongqing University of Technology, Chongqing, 400054, PR China.
Anal Chim Acta. 2025 Jul 1;1357:344069. doi: 10.1016/j.aca.2025.344069. Epub 2025 Apr 14.
The emergence of antibiotic-resistant microorganisms poses significant risks to public health. Therefore, the development of technologies capable of detecting antibiotics with high sensitivity and selectivity is essential for monitoring and controlling the spread of antibiotic resistance. Yet, current major available antibody-based antibiotic detection methods often face limitations in sensitivity, complexity, and cost, and commonly one target antibiotic can be detected in one assay.
On the basis of a three-way DNA junction (3-WJ) signal construct, we describe a multiplexed fluorescence resonance energy transfer (FRET) aptasensor strategy for highly sensitive simultaneous detection of sarafloxacin (SAR) and enrofloxacin (ENR) through cyclic DNAzyme and catalytic strand displacement reaction (CSDR) signal amplification cascades. Target antibiotics are recognized separately by the aptamers in DNAzyme/apamer duplexes to release active DNAzyme sequences, which cleave the dumbbell substrate hairpins to free ssDNAs to trigger subsequent CSDR between the assistance hairpins and the 3-WJ constructs for formation of many fluorophores 5-carboxyfluorescein (FAM)- and 2',7'-dimethoxy-4', 5'-dichloro-6-carboxyfluorescein (JOE)/6-carboxy-X-rhodamine (ROX)-labeled DNA duplexes. This leads to the pulling of FAM dye donor in proximity to the ROX and JOE dye acceptors, facilitating the yield of considerably amplified FRET signals at 555 nm and 605 nm for the SAR and ENR assays, respectively, with detection limits of 1.95 pM (0.76 ng/L) and 5.01 pM (1.8 ng/L) within 2.5 h. Additionally, this sensing method can selectively discriminate SAR and ENR against non-target antibiotics and has been validated for the simultaneous detection of SAR and ENR in milk samples.
Featured with the advantages of convenient and significant signal amplification capability as well as single excitation for multiplexed detection, the successful demonstration of our method for sensitive and simultaneous detection of two antibiotics therefore shows its promising potential for constructing different multiplexed aptasensors for detecting various low levels of biomolecules.
抗生素抗性微生物的出现对公众健康构成重大风险。因此，开发能够高灵敏度和高选择性检测抗生素的技术对于监测和控制抗生素抗性的传播至关重要。然而，目前主要的基于抗体的抗生素检测方法在灵敏度、复杂性和成本方面常常面临限制，并且通常一次检测只能检测一种目标抗生素。
基于三链DNA连接体（3-WJ）信号构建体，我们描述了一种多重荧光共振能量转移（FRET）适体传感器策略，用于通过环状脱氧核酶和催化链置换反应（CSDR）信号放大级联反应高灵敏度地同时检测沙拉沙星（SAR）和恩诺沙星（ENR）。目标抗生素分别被脱氧核酶/适配体双链体中的适配体识别，以释放活性脱氧核酶序列，该序列切割哑铃形底物发夹以释放单链DNA，从而触发辅助发夹与3-WJ构建体之间的后续CSDR，形成许多荧光团5-羧基荧光素（FAM）和2',7'-二甲氧基-4',5'-二氯-6-羧基荧光素（JOE）/6-羧基-X-罗丹明（ROX）标记的DNA双链体。这导致FAM染料供体靠近ROX和JOE染料受体，分别促进在555nm和605nm处为SAR和ENR检测产生显著放大的FRET信号，在2.5小时内检测限分别为1.95 pM（0.76 ng/L）和5.01 pM（1.8 ng/L）。此外，这种传感方法可以选择性地区分SAR和ENR与非目标抗生素，并且已被验证可用于同时检测牛奶样品中的SAR和ENR。
我们的方法具有方便且显著的信号放大能力以及用于多重检测的单激发的优点，因此成功展示了其用于灵敏且同时检测两种抗生素的潜力，显示出其在构建用于检测各种低水平生物分子的不同多重适体传感器方面的广阔前景。
