Jianguo Xu

Sensitive detection of foodborne pathogenic bacteria is essential for food safety monitoring and public health protection. Herein, we report a self-propagating hyperbranched rolling circle amplification (SP-HRCA) biosensing strategy enabled by a dual-function fluorogenic primer (DFP) for ultrasensitive detection of Salmonella. In this strategy, recombinase polymerase amplification (RPA) combined with λ-exonuclease (λ-exo)-assisted strand-selective digestion was employed to convert double-stranded DNA amplicons into target-specific single-stranded DNA intermediates, enabling efficient coupling between RPA and rolling circle amplification (RCA). More importantly, a specially engineered DFP was introduced as the central amplification relay unit. Upon hybridization with RCA products, the DFP simultaneously generated fluorescence signals through hairpin opening and functioned as a newly activated primer to initiate subsequent amplification events. This design transformed conventional fluorescent reporters from passive signal outputs into active amplification propagators, thereby intrinsically coupling signal transduction with amplification propagation. Through recursive amplification propagation, hyperbranched RCA nanostructures were continuously generated, resulting in markedly enhanced fluorescence output. Under optimized conditions, the proposed strategy exhibited a linear response from 101 to 106 cfu/mL with a detection limit of 8 cfu/mL. In addition, excellent specificity and satisfactory performance in practical food samples were achieved. This work provides a versatile strategy for constructing self-propagating nucleic acid amplification systems for ultrasensitive biosensing applications.

Antimicrobial peptides (AMPs) are promising candidates to combat antibiotic resistance; however, their application is limited by low targeting efficiency and in vivo stability. Here, we present a one‐pot, template‐free method for creating a supramolecular assembly of dendritic DNA nanostructures (DDN) and AMPs, enabling effective treatment of bacterial infections and wound healing. We produce DDN through terminal deoxynucleotidyl transferase‐mediated polyadenylation extension and hierarchical self‐hybridization, without a predesigned template. The pathogen‐specific aptamer‐functionalized DDN (aDDN) can be straightforwardly prepared, shows improved serum stability, effective protection of AMPs against enzymatic degradation, and demonstrates stronger in vitro antibacterial activity than AMPs alone. Using aDDN/AMP treatment, we achieved a 99% reduction in bacteria within the wound and nearly complete wound healing in 11 days, faster than with AMP alone. Notably, significantly denser collagen regeneration was observed, with no signs of systemic toxicity. This work will help translate AMPs into effective tools to fight bacterial infections and antimicrobial resistance.

Aflatoxin B₁ (AFB₁) is one of the most toxic and carcinogenic mycotoxins, and its trace-level determination in complex food matrices remains a major analytical challenge. Conventional chromatographic methods, while highly accurate, rely on expensive instrumentation and labor-intensive sample pretreatment, whereas most CRISPR/Cas-based biosensors depend on fluorescence intensity turn-on readouts that are vulnerable to matrix autofluorescence, photobleaching, and signal instability, limiting their reliability in real samples. Herein, we propose a rigidity-responsive fluorescence polarization (FP) biosensing strategy that integrates aptamer-based molecular recognition, rolling circle amplification (RCA), and CRISPR/Cas12a trans-cleavage for robust and matrix-tolerant AFB₁ detection. In this system, target binding induces the release of a complementary DNA strand from an immobilized aptamer duplex, initiating padlock probe circularization and RCA to generate abundant Cas12a-activating amplicons. A rationally engineered conformation-restricted depolarization reporter (CRD-Reporter), in which the fluorophore is confined within a rigid duplex framework, provides an intrinsically high FP signal. Upon Cas12a activation, collateral cleavage disrupts the rigid architecture, releasing freely rotating fragments and producing a pronounced FP decrease. Unlike intensity-based CRISPR assays, the FP readout effectively suppresses background interference and signal fluctuations. The proposed assay exhibits a wide linear range from 0.003 to 300 ng/mL with a low detection limit of 0.00113 ng/mL, high specificity, and excellent accuracy in grains, peanuts, and tea samples. This work establishes a robust FP-based CRISPR sensing paradigm for reliable mycotoxin monitoring in complex food systems.

Radiometal‐based positron emission tomography (PET) has become an indispensable tool for molecular imaging, enabling the noninvasive interrogation of biological processes across a broad range of time scales. Despite substantial advances in radionuclide production and targeting vector design, the in vivo performance and quantitative reliability of many radiometal PET tracers remain limited. These shortcomings are frequently attributed to biological factors; however, growing evidence indicates that they originate predominantly from unresolved challenges in coordination chemistry. This review critically examines commonly used PET radiometals, including [ ⁴⁴ Sc]Sc, [ ⁶⁴ Cu]Cu, [ ⁶⁸ Ga]Ga, and [ ⁸⁹ Zr]Zr, with a particular focus on the role of bifunctional chelating agents in governing metal stability, biodistribution, and imaging fidelity. Rather than providing a catalog of radionuclides and ligands, we identify recurring, cross‐system chemical constraints—such as incomplete metal coordination, insufficient kinetic inertness, and reliance on empirically inherited chelation frameworks—that continue to define the practical limits of radiometal PET. By comparing these systems across different biological time scales, this review highlights how chelator design, more than radionuclide physics alone, dictates translational success. Collectively, these insights underscore the need for mechanistically informed chelator development and standardized evaluation strategies to enable the next generation of robust, quantitative radiometal‐based PET imaging agents.

Salmonella is one of the most hazardous foodborne pathogens, posing a serious threat to public health and food safety worldwide. Conventional recombinase polymerase amplification (RPA)-CRISPR/Cas12a detection assays predominantly rely on the trans-cleavage of fluorescent reporters; however, such signal-generation modes are inherently susceptible to photobleaching, signal drift, and fluctuation, thereby compromising quantitative accuracy and long-term signal stability in practical pathogen detections. To overcome these limitations, we developed a trans-cleavage-independent fluorescence polarization (FP) sensing platform for the rapid and quantitative detection of Salmonella. Unlike conventional reporter-cleavage-based readouts, the proposed system exploits target-induced nucleoprotein assembly to achieve direct, physical signal amplification. In this design, a FAM-labeled forward primer serves as an intrinsic molecular reporter, while exonuclease I (Exo I) selectively degrades unincorporated primers, effectively suppressing background interference. Upon recognition of Salmonella genomic DNA, RPA produces rigid double-stranded amplicons that restrict fluorophore rotational freedom, and subsequent crRNA-guided Cas12a binding further increases molecular size and hydrodynamic volume, resulting in a stepwise enhancement of FP signals. The assay exhibits excellent linearity over a concentration range of 3 × 10¹–3 × 10⁶ CFU mL^–1, with an ultralow detection limit of 5 CFU mL^–1. In addition, it demonstrates outstanding photostability, reproducibility, and high specificity against nontarget bacteria. Importantly, reliable Salmonella detection was achieved in complex food matrices, including meat, eggs, and dairy products, with consistently high recoveries and strong tolerance to matrix interference, offering a promising alternative to conventional fluorescence-intensity-based CRISPR diagnostics in complex food systems.

DNA methyltransferases (MTases) play crucial roles in epigenetic regulation, and their abnormal activity is closely associated with various human diseases. Here, we report a customizable NAND logic-gate biosensing platform for highly sensitive and intelligent detection of DNA adenine methyltransferase (Dam MTase). An engineered methylation-CRISPR/Cas12a consensus sequence (MCCS, 5'-TTTGATC-3') was rationally designed to integrate the Cas12a PAM site, Dam methylation site, and DpnI recognition sequence into a unified functional motif. Coupled with a primer-triggered hybridization chain reaction (HCR), multiple tandem MCCS units were generated to amplify the fluorescence signal output. In this logic circuit, Dam, SAM, and DpnI serve as three biochemical inputs, and their combined presence ("1,1,1") yields a low-fluorescence "OFF" output according to the NAND logic rule. The system exhibited a broad linear detection range with an ultralow detection limit of 0.00032 U mL^-1, outstanding selectivity toward nontarget MTases, and satisfactory recoveries (98.16-100.03%) in human serum samples. Furthermore, it enabled quantitative evaluation of Dam inhibitors, revealing IC₅₀ values of 1.75 μM for 5-fluorouracil and 11.9 μM for penicillin G. This strategy provides a universal molecular computation-driven biosensing framework for enzyme activity analysis and inhibitor screening in complex biological systems.