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Enzyme-free fluorometric assay for chloramphenicol based on double stirring bar-assisted dual signal amplification
摘要： An enzyme-free fluorometric assay is described that accomplishes dual signal amplification by making use of a two stirring bars. Two Y-shaped DNA probes were designed and placed on the bars. When the target (with chloramphenicol as model analyte) is added, it triggers target recycling and simultaneously catalyzes hairpin assembly (CHA). A large fraction of DNA primers is released by the analyte from the bar to the supernatant and open hairpins with G-quadruplex DNA sequence. The G-quadruplex can specifically bind thioflavin T (ThT) to emit fluorescence (with excitation/emission maxima at 445 and 485 nm) for quantification of chloramphenicol. An enzyme is not needed. ThT is added to the system as a fluorescent DNA probe. All this strongly reduces the cost for sensor construction and usage. The dual signal amplification steps occur simultaneously which reduces the detection time. The assay was successfully employed to the determination of CAP in spiked milk and fish samples within 60 min and with a 16 pM limit of detection (at S/N = 3).
关键词： Food safety,Antibiotics detection,Thioflavin T,Catalyzed hairpin assembly,Target recycling
更新于2025-11-19 16:46:39
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Label-free optical biosensor for target detection based on simulation-assisted catalyzed hairpin assembly
摘要： Taking the advantage of the high selectivity of aptamers and enzyme-free catalyzed hairpin assembly (CHA) amplification strategy, we herein describe a label-free and enzyme-free sensitive fluorescent and colorimetric strategy for amplified thrombin detection in this paper. To support both biological inquiry and technological innovation, thermodynamic models are introduced to predict the minimum energy secondary structure of interacting nucleic acid strands and calculate the partition function and equilibrium concentration for complexes in our system. Then, the thermodynamics properties of interacting DNA strands and the reactions of toehold strand displacement-driven assembly have been simulated, validating the feasibility of the theory and optimizing the follow-up lab tests. Following that, our strategy for thrombin detection is proved to be feasible and effective in biological experiments.
关键词： Computation and simulation,G-quadruplex,catalyzed hairpin assembly,fluorescence biosensor,DNA strand displacement
更新于2025-09-10 09:29:36