DNA methylation is a crucial epigenetic modification whose abnormal alterations are closely associated with various tumors and are considered potential biomarkers for cancer diagnosis. However, achieving highly sensitive and selective detection of low-abundance methylated DNA in complex biological samples remains a significant challenge. This study developed a functionalized glass micropipette electrochemical sensing strategy based on the enzyme-responsive signal switching effect for highly sensitive detection of methylated DNA. This system utilizes glass micropipettes modified with polydopamine nanotubes (PDA-NTs) to construct an ion-transport interface, where the capture probe (cpDNA) is immobilized to achieve specific recognition of target sequences. The detection mechanism relies on sequence differences introduced by sodium bisulfite conversion and the selective cleavage action of the nucleic acid exonuclease Exo III: upon forming stable double-stranded structures with the cpDNA, methylated DNA triggers Exo III-mediated structural changes at the interface. This modulates ion transport behavior within the channels, generating a current response that switches from "Off" to "On", enabling effective differentiation between methylated and unmethylated DNA. This sensing system exhibits excellent linear response to methylated DNA within the 1 aM-100 fM range, with a detection limit as low as 8.5 aM, and enables reliable detection in 10% human serum samples. This strategy achieves sensitive detection through an enzyme-responsive signal switch without requiring PCR amplification or complex labeling procedures, providing a simple and effective method for rapid analysis of DNA epigenetic modifications.
DNA methylation is an important epigenetic biomarker for early disease screening and prognosis evaluation, but its reliable detection remains challenging because methylated DNA is often present at low abundance in complex biological backgrounds. Here, we report a methylation-sensitive bioelectronic sensing platform that integrates AciI-assisted target discrimination, CRISPR/Cas12a-mediated trans-cleavage, and vertical organic electrochemical transistors (vOECTs) amplification for highly sensitive methylated DNA detection. In this strategy, unmethylated DNA is selectively digested by AciI, while intact methylated DNA activates the crRNA-guided Cas12a system, triggering collateral cleavage of ssDNA reporters immobilized on the Au gate electrode. The resulting interfacial changes are efficiently amplified by the vOECTs through coupled electric-double-layer gating. The platform achieved quantitative methylated DNA detection from 100 fM to 100 pM with a sensitivity of 267.6 μA/dec and a detection limit of 100 fM. The sensor also exhibited good operational stability, reproducibility, and reliable recovery performance in artificial serum samples. This work demonstrates the potential of CRISPR/vOECTs bioelectronics for sensitive epigenetic analysis and presents a promising proof-of-concept for future non-invasive screening strategies.
Kun Xu, Sibo Wang, Kejie Zhang et al.· Talanta: The International J...· 0 citations
Aberrant DNA methylation has emerged as a critical epigenetic biomarker closely associated with bacterial pathogenicity and human tumorigenesis. In this study, a highly sensitive and label-free electrochemical biosensor has been developed for the detection of Dam MTase activity. 3'-aminated hairpin DNA probes (HP1) containing Dam MTase-specific recognition sites are immobilized onto the surface of a gold electrode. In the presence of Dam MTase, HP1 is methylated and subsequently cleaved by DpnI, thereby exposing free 3'-hydroxyl groups. These hydroxyl groups serve as primers and are extended by terminal deoxynucleotidyl transferase (TdT) to generate long poly-thymine (polyT) tails. As an electrochemical redox indicator with numerous positive charges, ruthenium hexaammine trichloride (RuHex) can be captured by negatively charged DNA extension products. Accordingly, a remarkably enhanced electrochemical signal is produced, and the signal response is proportional to methyltransferase activity. This biosensor exhibits a good linear relationship in the range of 0.1 U/mL to 20 U/mL, and the limit of detection (LOD) is as low as 0.57 U/mL. Furthermore, its application value has been verified through the successful screening of existing MTase inhibitors (5-fluorouracil and gentamicin), indicating its potential application prospects in clinical diagnosis and antibacterial drug screening.
Cancer incidence is high worldwide, and early treatment can reduce mortality rates. Circulating tumor DNA (ctDNA) serves as a biomarker for liquid biopsy, enabling early monitoring of tumor burden and treatment response. DNA nanobiosensors have become an important tool for detecting ctDNA due to their high efficiency and low cost. In this study, we developed a DNA lever nanomachine (DLN) based on a catalytic hairpin assembly (CHA)-driven conformational transition for the sensitive detection of ctDNA. The principle is to combine the CHA reaction with DNA lever-based mechanical amplification, enabling the detection of target ctDNA by monitoring changes in signal intensity. During the initial preparation stage, the nanomachine remains in a fluorescence-quenched state. When target ctDNA is present, CHA is activated, triggering downstream signal transduction and initiating a synergistic process of "target capture-conformational transition-signal amplification", ultimately leading to the restoration and amplification of the fluorescence signal. Additionally, by adjusting the fulcrum position, this machine enhances mechanical efficiency through an effort-saving lever mechanism. It minimizes internal driving force requirements by optimizing the torque load value at the load end, thereby further improving system responsiveness. The established fluorescent biosensing platform achieves satisfactory linear correlation with ctDNA concentrations ranging from 1 pM to 100 nM, with a limit of detection of 0.776 pM. This method offers a new strategy for CHA-based DNA mechanical fluorescence sensing and holds significant application potential in biological sample detection.
Sihui Wu, Q. Fu, Y. Lei et al.· ACS Sensors· 0 citations
Cellular signaling networks are orchestrated by complex interactions between gene expression and ion flux, yet tools for simultaneously visualizing these events in living cells remain limited. Herein, we report two orthogonal gold-nanoparticle-based DNA nanosensors that enable simultaneous imaging of sodium/hydrogen exchanger 1 (NHE1) mRNA and Na+ dynamics in hepatocellular carcinoma cells. The sensing mechanism relies on proximity-dependent fluorescence quenching. For mRNA detection, Cy3-labeled reporter strands hybridize to DNA-functionalized gold nanoparticles (AuNPs), holding the fluorophore close to the quenching surface. Target binding triggers strand displacement, releasing Cy3, and restoring emission. For Na+ detection, a Cy5-labeled substrate strand hybridizes with a Na+-specific DNAzyme anchored on AuNPs. Na+ activates the DNAzyme, cleaving the substrate at a defined site and liberating the Cy5 fluorophore. This dual-sensor system enables the quantitative monitoring of both analytes in living cells. Using this platform, we directly visualize that NHE1 mRNA downregulation suppresses intracellular Na+ accumulation, establishing a regulatory link between gene expression and ion homeostasis. Notably, mRNA-targeted hybridization inhibits cancer-cell migration in a dose-dependent manner, revealing a potential therapeutic mechanism. This approach provides a molecular tool for decoding ion-based signaling networks in cancer biology.
The integration of synthetic DNA receptors with metal–organic framework (MOF)-derived materials to construct synchronous dual-mode biosensors remains a challenging yet underexplored frontier. Herein, we report a novel DNA artificial mechanoreceptor (DAMR) engineered to enable synchronous fluorescence and colorimetric signal outputs upon target-induced mechanical actuation. In the fluorescence mode, the DAMR platform synergistically incorporates magnetic separation, DNA conjugate interface engineering, and a spatially confined catalytic hairpin assembly (CHA) as a mechanical switch for microRNA input. This switch exhibits tunable sensitivity and initiates dynamic DNA assembly via the release of fuel primers. The released fuel primers subsequently drive an autocatalytic assembly circuit (AAC), leading to fluorescence signal activation through fluorescence resonance energy transfer (FRET) within the hairpin probes. In the parallel colorimetric mode, target recognition triggers the hybridization-mediated assembly of a biotin-labeled complex on magnetic beads, which further recruits streptavidin-functionalized Fe–Co-MOF nanozymes. The Fe–Co MOF, exhibiting potent peroxidase-like activity, catalyzes the oxidation of TMB, generating a colorimetric signal whose intensity correlates with the target concentration. The uniqueness of this system stems from the strategic combination of magnetic beads, Fe–Co MOF nanozymes, and programmable DNA hairpin assemblies. This integration effectively mitigates matrix interference from proteases, eliminates the need for complex sample pretreatment, and enhances the overall biosensing robustness. Furthermore, the incorporation of MOF-derived materials with DNA receptors significantly improves the biostability and accelerates reaction kinetics. The modular design of DAMR also allows for the reprogramming of recognition sequences, extending the applicability of this method to diverse microRNAs and other nucleic acid targets. This work demonstrates a versatile and powerful approach for dual-signal transduction based on DNA mechanical receptors, holding considerable promise for advancements in mechanobiology, biosensor development, and biomedical diagnostics.
Jun Xu, Shanshan Tao, Bingshan Zhou et al.· Analytical Chemistry· 0 citations
Peptidylprolyl isomerase A (PPIA) catalyzes cis-trans isomerization of proline residues, a key process regulating protein folding and signal transduction. Its aberrant secretion is closely associated with tumor metastasis and progression, making it a promising biomarker. However, currently available immunoassays often suffer from insufficient sensitivity for low-abundance PPIA detection. Herein, we present an electrochemical biosensor that integrates self-propelled nanocarriers with DNA cascade amplification strategy for ultrasensitive PPIA detection. The biosensor employs PtNPs@COF particles that catalyze H2O2 decomposition to generate O2. This autonomous propulsion accelerates target binding in homogeneous solution and helps mitigate diffusion-limited binding kinetics. After PPIA binding, the liberated DNAzyme catalytically cleaves the co-immobilized substrate strands, generating numerous triggers for the catalytic hairpin assembly (CHA) reaction. The biosensor achieves a wide linear range from 1 pg/mL to 10 μg/mL with a detection limit of 0.330 pg/mL. It also exhibits excellent specificity and performs reliably in clinical lung adenocarcinoma serum specimens, demonstrating its promising applicability for early diagnosis of this malignancy.
Yu Hang, Lin Wang, Haojie Xie et al.· Biosensors & bioelectronics· 0 citations