Chronic lead exposure poses severe threats to human health, which demands a rapid detection strategy beyond conventional instrumentation-dependent approaches. While CRISPR/Cas12a systems offer promising alternatives through trans-cleavage activity, conventional Pb2+ biosensors relying on DNAzyme-generated intact activators suffer from high background signals due to interference from uncleaved substrates. To address this limitation, we developed a steric-hindrance-controlled activation strategy by employing a chimeric DNAzyme substrate (Sub) that prevents Cas12a binding until Pb2+-dependent cleavage occurs. This DNAzyme-mediated splitting releases two fragments (A1/A2) that rearrange into split activators, triggering the CRISPR/Cas12a trans-cleavage of a quenched reporter (6-FAM/BHQ1). Under the optimal condition, the sensor achieved a linear detection range of 2.5-25 μM (R2 = 0.998) with 2.18 μM LOD and high selectivity against interferents. Validation in tap water matrices demonstrated 98.6%-102.6% recovery (RSD 3.0%-7.5%), which showed robustness in real samples. This split-activator design paradigm eliminates background from uncleaved substrates without additional pretreatment steps to provide a versatile template for converting metal ions into CRISPR-detectable signals.

While CRISPR systems exhibit remarkable programmability in the field of nucleic acid editing, their extension to protein engineering faces a fundamental challenge, namely the traditional CRISPR tools lack the design to efficiently convert stimulus signals into the selective clustering of membrane receptors. This study develops a stimulus-responsive membrane-confined CRISPR-Cas12a platform that enhances selective clustering of membrane receptors for functional regulation. Specifically, a membrane-anchored DNA tetrahedral framework (TD-apt) was designed, which leverages vascular endothelial growth factor (VEGF) to activate Cas12a. Compared with unconfined CRISPR-Cas12a, membrane-confined CRISPR-Cas12a exhibits stronger cleavage activity, the interaction between the cellular-mesenchymal epithelial transition factor (c-Met) receptor and transferrin receptor (TfR) on A549 cells was efficiently modulated by nucleic acid assembly. This manipulation selectively inhibited c-Met function through spatial steric hindrance of TfR, modulating cellular behavior. Notably, the system’s generality was validated by engineering of c-Met homodimerization for activation. This cascading regulatory paradigm of environmental sensing (VEGF response)-nucleic acid computation (CRISPR-based nucleic acid molecular computation)-protein assembly (receptor topological remodeling) effectively extends CRISPR’s application boundaries to the field of non-genetic regulation protein-protein interaction (PPI) and establishes a versatile toolkit for dynamic and precise functional regulation.

Chronic wound healing remains challenging due to the risk of bacterial infection and the typical microenvironments characterized by oxidative stress, hypoxia, and insufficient angiogenesis. Herein, a hydrogel dressing (CLIHO) has been developed to efficiently promote the healing of methicillin-resistant Staphylococcus aureus (MRSA) infected wounds by integrating near-infrared (NIR)-controlled cascade reactions among hemin, indocyanine green (ICG), and l-arginine (l-Arg). Mild heat (approximately 45 °C) and cascade generation of reactive oxygen species (ROS), nitric oxide (NO), and ONOO− from the CLIHO hydrogel have been demonstrated under NIR irradiation. Benefiting from the O2 supplied by hemin-mediated decomposition of endogenous excess H2O2 in wounds, CLIHO hydrogel demonstrated hypoxia-alleviation-enhanced NO/phototherapy synergistic antibacterial activity, enabling it to effectively inhibit MRSA, Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Bacillus subtilis (B. subtilis) proliferation, as well as eradicate the formed biofilms. The antibacterial performance of the CLIHO hydrogel was significantly superior to that of the CIO hydrogel with phototherapy alone and the CLIO hydrogel without O2 supply. Upon NIR turn-off, the CLIHO hydrogel could remodel the microenvironment of chronic wounds by scavenging excessive ROS, reducing local H2O2 levels, and alleviating hypoxia while promoting angiogenesis by releasing trace amounts of NO under endogenous nitric oxide synthase. Importantly, compared with CLIO hydrogel (without hemin), the CLIHO hydrogel significantly accelerated the high-quality healing of MRSA-infected wounds by efficiently eliminating bacterial infection, balancing inflammatory responses, and promoting angiogenesis and collagen deposition. Hence, the prepared CLIHO hydrogel integrating NIR-controlled cascade reaction provides an efficient and secure dressing for accelerating healing of MRSA-infected wounds via hypoxia-alleviation-enhanced NO/phototherapy synergistic antibacterial and microenvironment remodeling.

The CRISPR/Cas12a system has emerged as a powerful tool for biosensing due to its high specificity, sensitivity and programmability. However, direct RNA detection is hindered by its inherent DNA-targeting trans-cleavage activity, which typically necessitates a reverse transcription amplification to convert RNA into DNA. Herein, we report a one-pot assay that enables direct detection of microRNA-21 without target amplification. The method employs a molecular switch probe (MSP) that recognizes miRNA-21 and activates Cas12a activity, along with an amplifier probe (AMP) that establishes a self-driven cascade signal amplification. This biosensor achieves a detection limit of 2.88 pM, with high accuracy (recovery of 95.5%-108.6%) and good precision (RSD: 2.35%-7.18%), and exhibits excellent specificity against homologous miRNAs. Using this assay, we successfully quantified the elevated levels of miRNA-21 in lung squamous carcinoma (H520) cells compared to normal bronchial epithelial cells (BEAS-2B). Furthermore, a methodological comparison with RT-qPCR revealed a similar trend between the two methods. This study provides a simple and reliable strategy for direct RNA detection using CRISPR/Cas12a.

Overexpression and abnormal activation of membrane proteins promote malignant tumor cell proliferation, while traditional interventions are often limited by drug resistance caused by target mutations. Here, we designed a stimulus-responsive DNA tetrahedron lysosome-targeted chimera (tFNA-LYTAC) strategy for degrading embrane proteins. Two tetrahedra are modified with trivalent HER2 aptamers (tFNA1-Apt) and trivalent IGF2R aptamers (tFNA2-Apt), enabling targeted binding to HER2 and IGF2R, and are then self-assembled into tFNA-LYTAC under VEGF stimulation to promote HER2 degradation. The trivalent aptamer modification strategy significantly enhances the overall binding capacity of tFNA1-Apt to cells. Importantly, tFNA-LYTAC formed only in the presence of VEGF, integrating tetrahedral multivalent aptamer targeting with stimulus responsiveness to improve the degradation specificity. VEGF-responsive tFNA-LYTAC promoted HER2 degradation and suppressed the phosphorylation of key downstream signaling proteins. The study innovatively proposes a “target recognition-stimulus response-endocytic degradation” mode that selectively regulates membrane proteins through programmable multivalent aptamer nanostructures.

MicroRNAs (miRNAs), as crucial regulators of gene expression, play significant roles in cancer development. Among them, miRNA-21 has been recognized as a promising biomarker for the diagnosis and prognosis evaluation of multiple cancers. However, existing CRISPR/Cas12a-based miRNA detection methods generally require reverse transcription and pre-amplification steps, which complicate the operational workflow and are prone to introducing cross contamination. Furthermore, the dependence on single-fluorescence signal detection makes them vulnerable to environmental and instrumental fluctuations, which results in risk of false-positive outcomes. Herein, we developed a ratiometric and sensitive CRISPR/Cas12a system with trans-cleavage-triggered feedback signal amplification for amplification-free detection of miRNA-21. The strategy employs a novel multifunctional switch probe that integrates target recognition, signal transduction, and cascade amplification within a unified system. A trans-cleavage-triggered feedback signal amplification circuit was constructed to enhance detection sensitivity without the need for additional enzyme systems. Moreover, a ratiometric fluorescence signal was implemented by calculating the fluorescence intensity ratio between CY5 and Auramine O, which effectively minimizes interference from environmental and instrumental variations and substantially improves detection accuracy and reliability. Experimental results demonstrate that the method achieves a detection limit of 1.28 nM, along with excellent specificity, accuracy, and reproducibility. In real sample analysis, differential expression levels of miRNA-21 in lung adenocarcinoma cells (A549) and non-cancerous adult bronchial epithelial cells (BEAS-2B) was observed, which highlights its potential value for clinical diagnostics.

Circulating tumor cells (CTCs) serve as critical biomarkers for diagnosis, treatment monitoring, and prognosis evaluation of lung cancer. However, their trace abundance in blood makes the development of integrated technologies for CTCs isolation, enrichment, and detection with both high specificity and sensitivity a key bottleneck for clinical translation. This study proposed a novel cascade cell-assisted enhanced fluorescence strategy that integrates dual-targeting recognition, cell-mediated cascade assembly, and enzymatic amplification. This dual-targeting strategy achieves efficient capture and enrichment of human non-small cell lung cancer cell line A549 cells through EpCAM antibodies, and uses dual-aptamer probe-mediated EGFR recognition to further improve detection specificity. Subsequently, cascade assembly of acute lymphoblastic leukemia CCRF-CEM cells around target cells combined with enzyme-catalyzed signal amplification constructed a highly sensitive fluorescence-based detection system, with a linear range of 10-104 cells/mL, a limit of detection (LOD) as low as 10 cells/mL, and recovery rates of 94.25%-105.41%. Importantly, clinical validation using whole blood samples from lung adenocarcinoma patients (n = 30) and healthy donors (n = 23) showed significant differentiation (P < 0.05, AUC of 0.728), underscoring its diagnostic potential. Moreover, its modular expandability allows extension to other tumor CTCs detection by replacing target recognition units.

The CRISPR/Cas12a system has emerged as a powerful tool for nucleic acid detection due to its high specificity, programmability and rapid reaction kinetics. However, the inherent incompatibility between target amplification and Cas12a-mediated detection in one-pot assays has limited its sensitivity, practicality and applicability in point-of-care testing (POCT). Recent advances in spatiotemporal regulation strategies have enabled precise control over the timing and localization of CRISPR/Cas12a reactions to address these limitations. This review comprehensively summarizes innovative approaches for temporal regulation including kinetic modulation, photoactivation and microfluidic architecture, as well as spatial regulation strategies such as micro-compartmentalization, molecular switches, immobilization techniques, and functionalized nanomaterials. These methodologies effectively decouple amplification and detection steps, minimize cross-contamination, enhance multiplexing capability, and extend detection scope to non-nucleic acid targets. Furthermore, we discuss emerging challenges related to system integration, practical deployment and standardization. Meanwhile, we offer perspectives on the future convergence of enzyme engineering, nanomaterial science, and artificial intelligence to advance the development of robust and field-deployable CRISPR/Cas12a sensors. This review aims to provide a foundational resource for researchers working toward the next generation of spatially and temporally optimized biosensors for clinical, environmental and industrial applications.

The management of infected chronic wounds is one of the urgent challenges. Herein, a hesperidin (Hes)-loaded self-assembled supramolecular hydrogel based on quaternized chitosan (CQHP) has been developed as an efficient photothermal antibacterial dressing for MRSA-infected wound healing. Specifically, CQHP hydrogels fabricated through the dynamic noncovalent interactions among CM-β-CD grafted QCS, Hes, proline and Fe3+, exhibited injectable and self-healing behaviors, along with adhesion, antioxidant, hemostatic and protein adsorption performance, satisfying the essential feature as chronic wound dressing. Of note, superior photothermal effect generated from the Hes-Fe3+ has been demonstrated, which endowed the CQHP hydrogels effectively and rapidly eliminate the E. coli, S. aureus and MRSA through photothermal therapy, thereby avoiding the use of antibiotics or photothermal conversion nanomaterials in hydrogels and substantially reducing the biological toxicity. Furtherly, sustained antibacterial performance in the absence of NIR can be achieved through the inherent antibacterial activities of Hes. Importantly, the developed CQHP hydrogels significantly promoted the closure of acute full-thickness scratch wounds, and exhibited remarkable better therapeutic effect on MRSA-infected wound than commercial 3M transparent film, by efficiency and sustained antibacterial activity, reducing inflammation, enhancing angiogenesis and collagen deposition, highlighting its promising application in the MRSA-infected wound healing with high efficiency, quality and security.

The identification of DNA methylation at specific sites is crucial for the early detection of cancer since DNA methylation is intimately associated to the occurrence and development of cancer. Herein, two types of sensors that can detect site-specific DNA methylation were developed to meet practical requirements using methylation sensitive restriction endonuclease and CRISPR/Cas12a. To accomplish rapid detection of target, an AciImediated CRISPR/Cas12a assay was developed by coupling AciI to recognize DNA methylation with Cas12a to identify site-specific DNA. Since protospacer adjacent motif (PAM)-dependent endonuclease activity and trans- cleavage activity of Cas12a, it is possible to detect site-specific DNA methylation within 2 h with high specificity and acceptable sensitivity. To satisfy the needs of trace target detection, we developed an GlaI-strand displacement amplification (SDA) assisted CRISPR/Cas12a system. The system converts double-stranded methylated DNA to abundant single-stranded by GlaI and SDA. Then, the combination of SDA and CRISPR/Cas12a enable cascades amplification of signal. The approach can therefore be used to detect methylation at different specified sites, even those without PAM, and can increase sensitivity with a detection limit down to 8.19 fM. Importantly, the assay can distinguish between colorectal cancer and precancerous tissue, as well as identify colorectal patients and healthy people. This study provides a new avenue for the development of new biosensors for methylation analysis, and the two methods devised have the potential to meet the multiple requirements of site-specific methylation testing in various clinical settings.

Exosomes that can cross the blood-brain barrier are promising biomarkers for glioma diagnosis, yet highly specific and sensitive detection of glioma-derived exosomes remains a challenge. Herein, a strategy of "proximity effect-mediated DNA self-assembly" has been proposed to achieve highly specific, sensitive, and flexible detection of glioma-derived exosomes. Exosomes are separated and enriched by CD63 aptamer-modified nanomagnetic beads; a pair of proximity probes simultaneously binds to PDPN and EGFR on the surface of exosomes, which will induce proximity effect-mediated DNA self-assembly with linker probes and the subsequent invertase-labeled signal amplification probes, thus converting one target exosome in the presence of multiple invertases. Benefiting from the dual signal amplification from nucleic acid self-assembly and enzymatic reaction, highly sensitive and flexible detection of glioma-derived exosomes can be achieved by using a portable blood glucose meter, with a limit of detection of 3 × 104 particles/mL. Of note, the combined detection of multiple exosomal surface markers (CD63/PDPN/EGFR) based on proximity hybridization significantly improves the specificity of glioma-derived exosome detection, enabling efficient discrimination of glioma cells from normal microglia and various other tumor cells. Furthermore, the level of CD63/PDPN/EGFR-positive exosomes in glioma patients was significantly higher than that of healthy subjects (P < 0.0001); compared with the CD63/PDPN- and CD63/EGFR-positive exosomes (AUCs of 0.852 and 0.895), the detection of CD63/PDPN/EGFR-based exosomes provides a remarkably accurate diagnosis of glioma (AUC of 0.98). Additionally, this strategy can be easily extended to the detection of other disease-derived exosomes just by replacing the corresponding recognition units.

Tumor-resident intracellular bacteria significantly impact cancer risk, pathology, prognosis, and treatment outcomes. However, existing probes for imaging low-biomass intracellular bacteria suffer from poor imaging contrast and susceptibility to interference from complex microenvironments. To tackle this, we designed activatable supramolecular fluorescent probes (ASFPs) that achieve high-contrast co-localization imaging of tumor-resident intracellular bacteria. Using S. aureus as a model, the probes specifically target its 16S rRNA, resulting in fluorescence enhancement mediated by beta-CD. Compared to monochromatic imaging, co-localization imaging of specific and conserved sequences within 16S rRNA contributes to enhance detection specificity and resistance to microenvironmental interference. Furthermore, in MCF-7 cell models and mouse xenografts harboring intracellular S. aureus demonstrated the efficacy of ASFPs for high-contrast imaging of tumor-resident bacteria at both cell and tissue levels.

Bacterial infection reduces quality of wound healing and even cause severe complications, but most current dressings adopt single antibacterial mode showed limited effectiveness. Herein, a plant extract quercetin (Que) composite chitosan/hyaluronic acid hydrogel (named CBCQ) with near-infrared (NIR) triggered controllable NO releasing was developed for efficient photothermal therapy/nitric oxide (PTT/NO) synergistic antibacterial and wound healing. Specifically, CBCQ hydrogel with chemical/physical crosslinked network was fabricated via host-guest interaction, metal coordination and Schiff base reaction. It exhibited porous structure, excellent injectability, self-healing, adhesion, antioxidant, and biocompatible properties suitable for wound dressings. Notably, superior PTT effect generated from Que-Fe3+ has been demonstrated, which served as a photothermal switch for the controllable NO release, thereby enable to utilize the physiological functions of NO at different concentrations, improving antibacterial efficiency by integrating PTT and NO benefits, and further allowing sustained antibacterial through Que when NIR was off. Importantly, CBCQ hydrogel achieved better healing of skin incision than commercial 3M medical glue and Film, and significantly improved the healing rate and quality of Methicillin-resistant Staphylococcus aureus (MRSA) infected wound by efficient and sustained antibacterial activity, inhibiting inflammation, and promoting collagen deposition and angiogenesis, highlighting its promising application in skin incisions and infected full-thickness skin wounds.

Combining imaging information from distinct types of molecular targets could offer profound insights into cellular functions and disease states, but remains challenging owing to poor compatibility of visualization labeling methods of multiple types of molecular targets and low imaging contrast. Here, we designed a surfaceenhanced fluorescence-encoded nanoprobe (SFENP) for in situ high-contrast imaging of gene expression in single cell. The application of beta-CD in SFENP has been demonstrated to enhance the fluorescence signals in two functional regions: a fluorescent encoding domain, loaded with six different ratios of fluorescein sodium (Flu) and Rhodamine B (RB) for qualitative analysis; and an activatable nucleic acid assembly domain that triggers the recovery of Cy5.5 fluorescence in the presence of targets for quantitative analysis. Furthermore, laser confocal imaging confirmed that SFENP enables in situ simultaneous imaging of three types of mRNAs (EGFR, VEGF, and PD-L1) and corresponding proteins to the three mRNA in single living cell, with enhancing imaging contrast and good compatibility. Concurrently, SFENP successfully achieved the monitoring of changes in EGFR mRNA and protein expression following siRNA interference. SFENP is anticipated to foster the advancement of high-contrast imaging methods and the implementation of molecular analysis studies across different target types in single cells.

The supramolecular nanoprobe could not only be activated by tumor cells and tissues to achieve high-contrast imaging of EGFR/EGFR and EGFR/HER2 dimers, but also successfully distinguish tumor cells and tissues from normal cells and tissues.

Diabetic wound repair is a global challenge due to bacterial infection and the typical microenvironments of hyperglycemia, high pH value, hypoxia, persistent inflammation and insufficient angiogenesis. Herein, a glucose-activated nitric oxide (NO) releasing and microenvironment regulation hydrogel dressing (COH-GB) has been developed for efficiently promoting infectious diabetic wound healing. Specifically, an artificial multienzyme nanoflower (GB nanoflower) composed of glucose oxidase (GOx) and hemoglobin (Hb) was synthesized and incorporated into the hydrogel formed by carboxymethyl chitosan (CMCS), oxidized sodium alginate (OSA) and hydroxyurea (HU). In which, the hyperglycemia-triggered cascade reaction could consume glucose, while providing hydrogen peroxide (H2O2) for the generation of NO by HU in the presence of Hb with peroxidase-mimicking activity. The COH-GB hydrogel with essential wound dressing characteristics could remodel the microenvironment of diabetic wounds by reducing local glucose and pH levels, alleviating hypoxia, and scavenging excessive ROS; and releasing NO under different blood glucose levels for antibacterial or angiogenesis. Importantly, the prepared COH-GB hydrogel significantly accelerated the MRSA-infected diabetic wound healing by effectively reducing wound infection, inhibiting inflammation, and promoting collagen deposition, angiogenesis and the migration and differentiation of fibroblasts and keratinocytes. Notably, the formation of GB nanoflowers could enhance the activity and stability of enzymes, and improve the cascade reaction efficiency, thereby inducing a more efficient performance in microenvironment regulation, antibacterial and angiogenesis, as well as wound healing. Hence, the prepared artificial multienzyme nanoflower composite hydrogel provides an efficient and secure dressing for promoting infectious diabetic wound healing via glucose-activated NO releasing and microenvironment regulation.

This review is dedicated to offering a new perspective on using DNA nanostructures to explore the progress of cell–cell communication.

Cell surface receptors play a key role in intracellular signaling, and their overexpression and activation are among the drivers of multiple diseases. Selective inhibition of cell surface receptors is important for regulating intracellular signaling pathways and cell behavior. Here, we design engineered aptamers to selectively inhibit receptor function. In this strategy, the aptamer specifically recognizing the extracellular structural domain of the EGFR, was conjugated to an adamantane moiety through linking arms of various lengths in order to obtain better performances toward EGFR. These interactions inhibit EGFR dimerization, thereby impeding the activation of downstream signaling pathways. It is shown that the adamantane-modified aptamers exhibit superior inhibition of downstream effector proteins relative to the unmodified aptamers. The optimal inhibitory effect was observed with a linker arm of 40 T-base in length. Notably, the best-performing adamantane-modified aptamer specifically binds to A549 cells with a dissociation constant (22.6 +/- 4.5 nM) that is approximately 4-fold lower than that of the parent EGFR aptamer (94.4 +/- 21.9 nM). We further combine the use of the adamantane-modified aptamer with that of genistein, a natural isoflavone compound with EGFR tyrosine kinase inhibition activity, to enhance the inhibitory effect on EGFR and its downstream signaling employing a synergistic action. This study is expected to provide a versatile approach for the improvement of existing aptamers obtaining increased selective inhibition of cell surface receptors.

This paper reports improved miR-21 detection signals based on a PGM combined with probe self-assembly and β-CD. The novel biosensor has been successfully applied to detect miR-21 in serum samples.

Tetracyclines (TCs) are the most commonly antimicrobial agents used in terrestrial food-producing animals. So, it is important to supervise tetracyclines residues in food for correcting the abuse of antibiotics in animals. In this paper, a novel label-free chemiluminescence (CL) assay without antibody was established for three TCs detection utilizing the catalytic ability of ferroferric oxide nanoparticles (Fe3O4 NPs). Fe3O4 NPs could facilitate the CL interaction between luminol and H2O2 due to their enzyme-like activity. Interestingly, TCs could enhance the catalytic ability of Fe3O4 NPs and result in a further amplification of the CL intensity. Under optimal conditions, the CL intensity varied linearly with the concentration of tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), and ranging from 10~2400, 10~2800, and 5~2100 nmol/L, respectively; The limits of detection were 4 nmol/L for TC, 6 nmol/L for OTC, and 2 nmol/L for CTC. This CL strategy was applied successfully in testing three TCs residues in milk, eggs and honey samples with more sensitive results than that of the colorimetric, fluorescence and high-performance liquid chromatography (HPLC) methods, which provided an alternative strategy for monitoring the correct use of TCs.