In
this work, a novel ratio electrochemical biosensing platform
based on catalytic hairpin assembly target recovery to trigger dual-signal
output was developed for ultrasensitive detection of microRNA (miRNA).
To achieve the ratiometric dual-signal strategy, methylene blue (MB),
an electrochemical indicator, was ingeniously loaded into the pores
of graphene aerogel (GA) and metal–organic framework (MOF)
composites with high porosity and large specific surface area, and
another electrochemical indicator Fe-MOFs with distinct separation
of redox potential was selected as a signal probe. Concretely, with
the presence of the target miRNA, the CHA process was initiated and
the signal probe was introduced to the electrode surface, producing
abundant double-stranded H1-H2@Fe-MOFs-NH2. Then, the measurement
and analysis of the prepared ratiometric electrochemical biosensor
by differential pulse voltammetry (DPV) showed that the introduction
of the target miRNA led to an increase in the oxidation peak signal
of Fe-MOFs (+0.8 V) and a decrease in the oxidation peak signal of
MB (−0.23 V). Therefore, the peak current ratio of I
Fe‑MOFs/I
MB could be employed to accurately reflect the actual concentration
of miRNA. Under optimal conditions, the detection limit of the proposed
biosensor was down to 50 aM. It was worth noting that the proposed
biosensor exhibited excellent detection performance in a complex serum
environment and tumor cell lysates, showing great potential in biosensing
and clinical diagnosis.
DNA methylation is considered as a potential cancer biomarker. The evaluation of DNA methylation level will contribute to the prognosis and diagnosis of cancer. Herein, we propose a novel assay based on endonuclease-assisted protospacer adjacent motif (PAM)-free recombinase polymerase amplification coupling with CRISPR/Cas12a (E-PfRPA/Cas) for sensitive detection of DNA methylation. The methylation-sensitive restriction enzyme (MSRE) is first used to selectively digest unmethylated DNA, while the methylated target remains structurally intact. Therefore, the methylated target can initiate the RPA reaction to generate a large amount of double-stranded DNA (dsDNA). To avoid the dependence of PAM site of CRISPR/Cas12a, one of the RPA primers is designed with 5′phosphate terminuses. After treating with Lambda, the sequence with 5′-phosphate modification will be degraded, leaving the singlestranded DNA (ssDNA). The CRISPR/Cas12a can accurately locate ssDNA without PAM, then initiating its trans-cleavage activity for further signal amplification. Meanwhile, non-specific amplification can be also avoided under Lambda, effectively filtering the detection background. Benefiting from the specificity of MSRE, the high amplification efficiency of Lambda-assisted RPA, and the self-amplification effect of CRISPR/Cas, the E-PfRPA/Cas assay shows outstanding sensitivity and selectivity, and as low as 0.05% of methylated DNA can be distinguished. Moreover, the lateral flow assay is also introduced to exploit the point-of-care diagnostic platform. Most importantly, the proposed method shows high sensitivity for determination of genomic DNA methylation from cancer cells, indicating its great potential for tumor-specific gene analysis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.