Ochratoxin A (OTA) is a compound of concern due to its potential health effects on humans. Detecting OTA in food is crucial for safeguarding public health. In this study, we fabricated a multi-DNAzyme cascade reaction-mediated colorimetric aptasensors for OTA detection, integrating autocatalytic Mg 2+ -dependent DNAzyme cleavage (MNAzyme) and an entropy-driven circuit. In brief, the recognition between the aptamer and target OTA led to the release of DNA1. Subsequently, DNA1 hybridized with DNA2, generating an upstream MNAzyme that facilitated the production of a downstream MNAzyme. These MNAzymes possess similar substrate binding arms, enabling them to catalyze the same substrate. The catalytic efficiency of MNAzymes towards the substrates was enhanced due to the increasing concentration of MNAzymes. The cleavage products then triggered an entropy-driven cycle to generate a signal. Under optimal conditions, the sensing system exhibited low detection limits of 48.97 fM for OTA. Additionally, the proposed aptasensor was successfully applied to quantitatively analyze OTA in food samples. Thus, the multi-DNAzyme cascade reaction-mediated colorimetric aptasensors offer an adaptable platform for detecting traces of OTA contaminant in food.
Single miRNAs are not specific and accurate enough to meet the strict diagnosis requirements in practice. Therefore, simultaneous monitoring of multiplexed miRNA in biological samples can not only improve the accuracy and specificity of bioassays but also avoid the squandering of valuable biological specimens. Herein, we designed a metal sulfide nanoparticle-based dual barcode-triggered DNAzyme cascade strategy for the sensitive and simultaneous multiplex miRNA detection in a single assay. Firstly, the capture probes (H1, H2) specifically recognize targets (miRNA-21, miRNA-141), exposing the stem of H1 and H2. Then, with the introduction of a detection probe (CuS-H3, ZnS-H4), the exposed H1 and H2 catalyze the hairpin assembly (CHA) reaction, realizing target miRNA recycling, and forming H1/H3-CuS and H2/H4-ZnS complexes. Subsequently, the formed H1/H3-CuS and H2/H4-ZnS complexes are encoded on magnetic beads through the biotin/streptavidin interaction. The CuS and ZnS nanoparticles captured by magnetic beads release thousands of Cu2+ and Zn2+via the cation exchange reaction. Finally, the released Cu2+ and Zn2+ specially activate the DNAzyme of the catalytic and molecular beacon (CAMB) system. The CAMB system affords an amplified fluorescence signal output by cycling and regenerating the metal ion-dependent DNAzyme to realize multiple enzymatic turnovers. Benefiting from target recycling, nanoparticle amplification, and catalytic and molecular beacon amplification, there is substantial amplification and the target miRNAs can be detected at 0.06 fM (miRNA-21) and 0.048 fM (miRNA-141) in a single assay. Furthermore, the high selectivity and accuracy of the assay were proved by practical analysis of different cancer cells, which exhibited good practicability in multiplex miRNA detection in clinical sera. The results indicate that the proposed strategy holds great potential for the sensitive detection of multiplex cancer biomarkers and offers the opportunity for future applications in clinical diagnosis.
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