A fluorescence chip based on a membrane-anchored, bipedal DNAzyme walker assembled on small extracellular vesicles (sEVs) surfaces via catalytic hairpin assembly (CHA) opens a new paradigm for designing efficient DNA nanomachines on membrane-enveloped targets.
Abstract
We report a fluorescence chip based on a membrane-anchored, bipedal DNAzyme walker assembled on small extracellular vesicles (sEVs) surfaces via catalytic hairpin assembly (CHA). Unlike previously reported single-arm or proximity-ligation-dependent walkers on nanoparticle supports, this CHA-triggered DNAzyme walker is designed with a bipedal architecture and operates directly on the sEVs membrane. In our design, CHA simultaneously generates two DNAzyme arms on the same sEVs, enabling faster substrate cleavage kinetics and superior signal amplification. The assay achieves a theoretical detection limit of 2.8 particles/μL, approximately 1.5-fold lower than a comparable single-arm architecture. Dual recognition of the sEVs lipid bilayer and surface EpCAM protein (using a cholesterol-modified substrate and an anti-EpCAM antibody) effectively eliminates false-positive signals from free EpCAM or soluble interferents. The entire reaction cascade is integrated onto a chip platform, improving reproducibility and point-of-care potential. The chip performs robustly in fetal bovine serum and clinical serum samples, with recovery rates of 99.1-103.8% and strong correlation with nanoparticle tracking analysis. By establishing a CHA-assisted, bipedal walking mechanism on biological membranes, this work opens a new paradigm for designing efficient DNA nanomachines on membrane-enveloped targets.
The development of highly sensitive and reliable biosensors is essential for the early diagnosis and therapeutic monitoring of cancer. Herein, we report a dual-mode fluorescence-colorimetric biosensing platform for the ultrasensitive detection of miRNA-21, integrating AuNP-supported dual-stage rolling circle amplification (RCA) with an RCA-generated multipedal binding-and-cleavage mechanism. The sensing hierarchy is initiated by target-dependent activation of the primary RCA on the gold nanoparticle (AuNP) surface, producing long DNA strands with multiple repeated recognition domains that function as numerous walker "legs" and form a DNA-rich nanogel matrix. Through these repeated domains, the RCA-generated multipedal walker repeatedly and multivalently binds fluorophore-labeled hairpin substrates on the AuNP surface, thereby facilitating APE1-mediated substrate cleavage and fluorescence recovery. The APE1-generated cleavage fragments subsequently initiate the secondary RCA following the addition of the T2 template. The two successive RCA reactions ultimately produce an Au@RCA DNA nanogel containing abundant G-quadruplex/hemin DNAzymes that catalyze a visually detectable color change. By integrating the multipedal walker-mediated multivalent binding-and-cleavage process with dual-stage RCA, the platform enables dual-mode fluorescence-colorimetric detection with efficient signal amplification and low background, achieving limits of detection of 9.854 and 790 fM for the fluorescence and colorimetric modes, respectively. Furthermore, the two complementary outputs provide independent signal readouts for cross-validation. The biosensor demonstrated excellent specificity and distinguished the endogenous responses of MCF-7, HeLa, and HEK293T cell lysates. These results support the potential of the platform for ultrasensitive tumor-associated biomarker detection, while the cell-lysate experiments represent a preliminary proof-of-concept evaluation in complex biological matrices.
Hongqun Yang, Long-Hao Li, Mu Niu et al.· ACS Applied Materials and In...· 0 citations
This work presents a programmable molecular device that integrates Boolean logic computation with spatial confinement to overcome limitations in affinity, specificity, and off-target binding, and demonstrates a strategy for programming high-fidelity molecular interactions on interested cell surfaces.
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
Fluorescent light-up aptamers (FLAPs) are attractive signaling tools due to their label-free fluorescence and programmability. However, DNA-based FLAP sensors are often constrained by low fluorogenic activation efficiency, limited intrinsic target responsiveness, and insufficient signal amplification. In this study, we integrated a DNA-based FLAP (DAP) with a three-branched DNA (TBD) scaffold to construct a multivalent FLAP nanostructure (DAP-TBD) that exhibited enhanced fluorogenic activation toward Auramine O (AO). Compared with monovalent DAP, the trivalent DAP-TBD showed a 3.3-fold increase in fluorogenic activation efficiency, indicating a clear multivalent effect. The system adopts a modular architecture in which DAP functions as a replaceable FLAP model. Using a target-triggered catalytic DNA assembly (CDA) circuit composed of three rationally designed hairpin DNAs, multiple DAP-TBD nanostructures are assembled isothermally, enabling target-triggered signal generation and amplification without the need for labeled probes. The CDA-based DAP-TBD system selectively detects target single-stranded DNA with a detection limit of 235 pM. Furthermore, coupling the DAP-TBD system with an exonuclease-assisted hairpin and an aptazyme enabled label-free detection of microRNA and adenosine triphosphate, demonstrating the versatility of the system as a signal sensor that can interface with diverse upstream transducer modules. The system requires no fluorophore or quencher labeling, relies on only three unmodified DNA strands, and offers programmability, modularity, and cost-effectiveness. Overall, this study presents a multivalent DNA-based FLAP nanostructure and provides a versatile strategy for developing label-free fluorescent biosensors.
Yao Zhang, Xiao Liu, Hong-Liang Ma et al.· Talanta: The International J...· 0 citations
The reliable electrochemical detection of immunoglobulin G (IgG) in complex biological fluids is severely hampered by nonspecific protein fouling and enzymatic degradation. Inspired by natural lasso peptides, we engineer a multifunctional lasso-like peptide (LaP) composed entirely of D-amino acids that uniquely integrates a terminal cysteine anchoring domain, a zwitterionic antifouling segment, and a specific IgG recognition segment into a single topology-constrained architecture. The covalently closed lasso topology sterically restricts protease access to cleavage-sensitive peptide bonds, while the all-D-amino acid backbone is inherently invisible to naturally occurring proteases. Together, these features provide dual physical and stereochemical protection while preserving the conformational freedom of the recognition domain for efficient target binding. Compared with a branched peptide (BrP) of identical sequence, the LaP-modified interface exhibits markedly superior resistance to nonspecific protein adsorption in undiluted serum. Molecular dynamics simulations reveal that the lasso constraint induces a superficial, low-affinity binding pose toward carboxypeptidase Y (CPY), whereas BrP penetrates deeply into the enzyme active pocket. Consequently, the LaP-based biosensor shows negligible signal change after CPY treatment, whereas BrP loses most of its activity. The LaP/AuNPs/PANI electrochemical biosensor achieves excellent selectivity and maintains its analytical performance even in complex serum matrices. This work establishes a paradigm for designing topology-constrained, multifunctional peptide interfaces that overcome the longstanding trade-off between biofouling and enzymatic degradation, opening new avenues for high-fidelity electrochemical biosensing in complex biological matrices.
This work introduces DNA-FLASH (DNA-based FLuorescence Amplification upon Single-target Hybridization), a DNA nanosensor concept for digital biosensing that may support the development of next-generation biosensors capable of addressing pressing global challenges, including rapid disease detection, environmental sustainability, and personalized healthcare.
