The monitoring and control of mycotoxins has caused widespread concern due to their adverse effects on human health. In this research, a simple, sensitive and non-label fluorescent aptasensor has been reported for mycotoxin ochratoxin A (OTA) detection based on high selectivity of aptamers and amplification of non-enzyme hybridization chain reaction (HCR). After the introduction of OTA, the aptamer portion of hairpin probe H1 will combine with OTA to form OTA-aptamer complexes. Subsequently, the remainder of the opened H1 will act as an initiator for the HCR between the two hairpin probes, causing H1 and H2 to be sequentially opened and assembled into continuous DNA duplexes embedded with numerous G-quadruplexes, leading to a significant enhancement in fluorescence signal after binding with N-methyl-mesoporphyrin IX (NMM). The proposed sensing strategy can detect OTA with concentration as low as 4.9 pM. Besides, satisfactory results have also been obtained in the tests of actual samples. More importantly, the thermodynamic properties of nucleic acid chains in the monitoring platform were analyzed and the reaction processes and conditions were simulated before carrying out biological experiments, which theoretically proved the feasibility and simplified subsequent experimental operations. Therefore, the proposed method possess a certain application value in terms of monitoring mycotoxins in food samples and improving the quality control of food security.
Herein, we designed a label-free fluorescent aptasensor based on triple-helix DNA and G-quadruplex for carbohydrate antigen (CA15-3) detection. Triplex-helix structure can be formed with Insert G-rich DNA (IG) and Aptamer DNA (Apt), which likes a "lock" and the G-rich sequences are locked. The CA15-3 was the "key", which specifically combined with aptamer sequences of Apt, causing IG liberated from the triplex-helix "lock". Hereafter, the G-rich sequences of IG were formed into G-quadruplex and specifically interacted with N-methylmesoporphyrin IX (NMM), which greatly enhanced fluorescence of the solution. However, when the "key" was not existing, the "lock" would be fastened and fluorescence intensity didn't change. According to this proposal, the concentration of CA15-3 can be effectively detected from 0.01 U mL-1 to 5 U mL-1 with a detection limit (LOD) of 0.01 U mL-1. Furthermore, this proposal can be applied to spiked human serum with great precision and reproducibility.
Background:
Molecular logic gate always used fluorescent dyes to realize fluorescence signal. The labeling of the fluorophore is relatively expensive, low yield and singly labeled impuritiesaffects the affinity between the target and the aptamer. Label-free fluorescent aptamer biosensor strategy has attracted widespread interest due to lower cost and simple.
Objective:
Herein, we have designed a AND logic gate fluorescent aptasensor for detecting carbohydrate antigen 15-3(CA15-3) based on label-free fluorescence signal output.
Materials and Methods:
A hairpin DNA probe consists of CA15-3 aptamer and partly anti-CA15-3 aptamer sequences as a long stem and G-rich sequences of the middle ring as a quadruplex-forming oligomer. G-rich sequences can fold into a quadruplex by K+, and then G-quadruplex interacts specifically with N-methylmesoporphyrin IX(NMM), leading to a dramatic increase in fluorescence of NMM. With CA15-3 and NMM as the two inputs, the fluorescence intensity of the NMM is the output signal. Lacking of CA15-3 or NMM, there is no significant fluorescence enhancing, and the output of the signal is “0”. The fluorescence signal was dramatically increasing and the output of the signal is “1” only when CA15-3 protein and NMM were added at the same time.
Results:
This biosensor strategy possessed selectivity, high sensitivity for detecting CA15-3 protein from 10 to 500 U mL-1 and the detection limit was 10 U mL-1, and also showed good reproducibility in spiked human serum.
Conclusion:
In summary, the proposed AND logic gate fluorescent aptasensor could specifically detect CA15-3.
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