Aptazyme is a chimera of functional nucleic acids , which integrates recognition and amplification elements to simplify the assay process and improve sensing efficiency. However, its application may be limited by signal leakage. In this work, we innovatively integrate the AβO aptamer and an MNAzyme (multicomponent nucleic acid enzyme) for highly efficient detection of AβO. The aptamer and half of the MNAzyme are positioned at one strand, and the other half of the MNAzyme is integrated with a toehold sequence. These two sequences cannot hybridize to activate the MNAzyme until the target is added. The background signal is significantly reduced by the split format and the secondary structure of DNA probes formed in the absence of the target. The proposed aptazyme can not only achieve amplification through enzymatic catalysis but also greatly improve the efficiency of signal transduction and output. We systematically investigated the influence of different DNA probes on the detection performance, and the optimized aptazyme can detect as low as 26.5 pM targets in 1h. The stability of this method was also investigated by detection targets in real biological samples.
Alzheimer's disease (AD) is a progressive and irreversible neurological disorder characterized by degeneration or dysfunction of central neurons. Recent studies reveal that soluble β-amyloid oligomers (AβO) serve as more reliable biomarkers for early AD detection. To address this issue, we specifically designed a dual-mode signal nanopipette sensor for AβO detection. The electrochemical signal is generated by ferrocene (Fc), while the fluorescence signal is based on Cy5. Upon capturing AβO, the double-stranded DNA aptamer (Apt1/DNA2) initiates a dual-signal process involving hybridization chain reaction (HCR). This mechanism promotes the formation of novel double-stranded DNA structures and activates the auxiliary cleavage function of the CRISPR/Cas12a system. In the presence of AβO, the fluorescence response exhibits linear correlation with AβO concentration (R 2 = 0.99557). The combined specificity of the double-stranded aptamer and CRISPR/Cas12a, coupled with the dual signal provided by HCR, significantly enhances the system's specificity and sensitivity. The detection method achieves a minimum detection limit of 2.15 pM with a linear detection range of 0.1–120 nM. Furthermore, this highly specific and sensitive system not only paves the way for detecting biomarkers of other diseases but also offers a highly promising research direction.
Xiao-Hong Wen,Xue-Feng Zhao,Xiao-Huan Wang,Yang Wang,Jing-Chun Guo,Hou-Guang Zhou,Chuan-Tao Zuo,Hong-Liang Lu
Amyloid β protein oligomer (AβO) has been found to be a crucial biomarker of Alzheimer’s disease (AD). Exploring an accurate detection method of AβO is of great significance for early AD diagnosis and treatment. Herein, a fluorescent biosensor for AβO detection is designed. It integrates fluorescence resonance energy transfer (FRET)-based fluorescence detection with a poly(dimethylsiloxane) (PDMS)-based microfluidic chip. The carboxyl fluorescein-modified AβO aptamers are employed as the fluorophore, and three-dimensional Fe3O4/MXene nanospheres are used as the fluorescence quencher for the first time. Moreover, the PDMS-based multichamber microfluidic chip serves as a fluorescence detection platform. The Fe3O4/MXene-based nanosphere-based biosensor shows an excellent linear relationship between the fluorescence intensity and the logarithm of the AβO concentration in the range of 0.10–200 nM. The detection limit is ∼0.05 nM at the ultralow sample volumes of ∼4.50 μL. These results strongly show that the fabricated high-performance biosensor can be widely used in smart healthcare including but not limited to medical diagnosis, health monitoring, and biological studies.
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