Aptamer-involved fluorescence amplification strategy facilitated by directional enzymatic hydrolysis for bioassays based on a metal-organic framework platform: Highly selective and sensitive determination of thrombin and oxytetracycline

The authors describe a fluorescence amplification strategy for selective and sensitive fluorescent assays based on aptamer-triggered directional hydrolysis and on the use of metal organic frameworks (MOFs) of type MIL-101. The method is implemented by mixing MIL-101, fluorescein-labeled DNA probes, exonuclease of type RecJf, and targets. A smart design of the three-adenine bulge on the DNA probe facilitates exonuclease-assisted directional hydrolysis, making the strategy universal for determination of both proteins and small molecules as well. Good selectivity is accomplished due to the use of MIL-101 protected aptamers, while high sensitivity resulted from exonuclease-assisted target-recycling signal amplification. The power of the method is demonstrated by analyzing the two model analytes thrombin (a fairly large protein) and oxytetracycline (OTC; a small molecule antibiotic). The limits of detection are 15 pM for thrombin and 4.2 nM for OTC. This is two orders of magnitude lower than that of conventional 1:1 homogeneous fluorescence assays. The strategy was successfully applied to the analysis of thrombin and OTC in real samples. In our perception, the strategy presented here has a wide scope for selective and sensitive detection of trace analytes for which appropriate DNA probes can be identified. Schematic of an aptamer-triggered amplification strategy for sensitive bioassays based on fluorescence quenching of a MOF and 5′→3′ directional hydrolysis of RecJf exonuclease. The adenine-bulge on the DNA probe facilitates exonuclease hydrolysis, making the strategy universal for both proteins and small molecules detection.