In this work, an enzyme-free biosensor
is reported for mycotoxin
detection based on a toehold-mediated catalytic hairpin assembly (CHA)
and a DNAzyme-cascaded hydrolysis reaction. In the presence of a mycotoxin,
the recognition between an aptamer and the mycotoxin releases the
trigger DNA. The trigger DNA initiates the toehold-mediated CHA, generating
large amounts of partial duplex B/C with four toeholds, which can
be used to assemble the DNAzyme-cascaded hydrolysis reaction. Furthermore,
through a collaborative autoassembly reaction among the B/C duplex,
DNA1, and DNA2, supramolecular nanostructures corresponding to Mg2+-dependent DNAzymes can be formed. With the incubation of
Mg2+, the dual-modified (TAMRA/BHQ2) substrate strand DNA2
will be cleaved into two fragments, yielding a high TAMRA fluorescence
signal for mycotoxin testing. Under optimal conditions, the sensing
system was ultrasensitive and showed low detection limits of 0.2 pM
for ochratoxin A (OTA), 0.13 pM for aflatoxin B1 (AFB1), and 0.17
pM for zearalenone (ZEN). The mycotoxin aptasensor also exhibited
high selectivity and was successfully applied for the quantitative
analysis of OTA, AFB1, and ZEN in wine samples. Due to the advantages
of flexibility and versatility, this mycotoxin platform was used to
fabricate several concatenated logic gates including “AND–INHIBIT”,
“INHIBIT–OR”, “OR–AND”,
and “OR–INHIBIT” logic biocomputings. Such multiple
functions of the logic system provided a universal sensing strategy
for the intelligent detection of multiplex mycotoxins, demonstrating
considerable potential in food safety and environmental monitoring.
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