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In this study, we developed an aptamer-based fluorescent sensing platform for the detection of ochratoxin A (OTA) based on RecJf exonuclease-assisted signal amplification and interaction between graphene oxide (GO) and the OTA aptamer (OTA-apt). After optimizing the experimental conditions, the present aptamer-based sensing system can exhibit excellent fluorescent response in the OTA assay, with a limit of detection of 0.07 ng/mL. In addition to signal amplification, this strategy is also highly specific for other interfering toxins. Furthermore, this aptasensor can be reliably used for assessing red wine samples spiked with different OTA concentrations (2.4, 6 and 20 ng/mL). The proposed assay plays an important role in the field of food safety and can be transformed for detecting other toxins by replacing the sequence that recognizes the aptamer.

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. Graphical abstract 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.

Ochratoxin A (OTA) is a mycotoxin widely found in foodstuffs such as cereal grains. It greatly threatens human health owing to its strong toxicity and high stability. Aptasensors have emerged as promising tools for the analysis of small molecule contaminants. Nucleic-acid-based signal amplification enables detectable signals to be obtained from aptasensors. However, this strategy often requires the use of complex primers or multiple enzymes, entailing problems such as complex system instability. Herein, we propose a fluorescent aptasensor for the ultrasensitive detection of OTA in cereal products, with signal amplification through RecJf exonuclease-assisted target recycling. The aptamer/fluorescein-labeled complementary DNA (cDNA-FAM) duplex was effectively used as the target-recognition unit as well as the potential substrate for RecJf exonuclease cleavage. When the target invaded the aptamer-cDNA-FAM duplex to release cDNA-FAM, RecJf exonuclease could cleave the aptamer bonded with the target and release the target. Thus, the target-triggered cleavage cycling would continuously generate cDNA-FAM as a signaling group, specifically amplifying the response signal. The proposed exonuclease-assisted fluorescent aptasensor exhibited a good linear relationship with OTA concentration in the range from 1 pg/mL to 10 ng/mL with an ultralow limit of detection (6.2 ng/kg of cereal). The analytical method showed that recoveries of the cereal samples ranged from 83.7 to 109.3% with a repeatability relative standard deviation below 8%. Importantly, the proposed strategy is expected to become a common detection model because it can be adapted for other targets by replacing the aptamer. Thus, this model can guide the development of facile approaches for point-of-care testing applications.