Yiping Chen

Rapid and sensitive detection of pathogenic bacteria remains challenging due to insufficient signal gain and uncontrollable signal transduction strategies that are easily perturbed by environmental fluctuations. Here, we present a micro-orifice resistance biosensor that converts a molecular circuit-programmed aggregation–dispersion transition into a quantifiable electrical signal. Entropy-driven circuit acts as the core regulatory module, maintaining signal probes in a stable low-entropy aggregated state while providing a built-in, target-responsive pathway for controlled dispersion. Upon recognition of bacterial DNA, the molecular circuit shifts toward a higher-entropy dissociated state, releasing dispersed microparticles that generate distinct resistance pulse signatures. To enhance initiator availability and strengthen the molecular circuit activation, a hairpin-free bridge-hybridization chain reaction (nHCR) supplies complementary upstream amplification without relying on enzymatic processes. The sensor achieves a limit of detection of 27 CFU/mL for Listeria monocytogenes in an amplification-free manner, comparable to quantitative PCR in real sample analysis. Our molecular circuit-programmed dispersion switching, supported by dual amplification, offers a robust and scalable strategy for rapid pathogen analysis.

The 5′-repeat fragments released during pre-crRNA maturation are critical yet understudied components of the CRISPR/Cas12a system. Here, we demonstrate that engineered 5′-repeat fragments can potently activate Cas12a cleavage, with efficiency strongly dependent on the length of the 3′-spacer. Strikingly, complete truncation of the 3′-spacer generates a “chiral-like crRNA” conformation that induces a delayed-switch mode of Cas12a activation, fundamentally distinct from conventional mature crRNA. Leveraging this characteristic, we develop the delayed cleavage feature-mediated one-pot sensing strategy that resolves the long-standing challenge of incompatibility between Cas12a-based cleavage reaction and nucleic acid amplification, achieving a 1000-fold improvement in sensitivity over that of the conventional mature crRNA-mediated one-pot method. Furthermore, we integrate a cleavage-based one-pot assay with a portable temperature-controlled fluorescence imaging device to create an on-site diagnostic platform for high-throughput screening. Our study further advances the understanding of the crRNA-guided mechanism and facilitates the expansion of its applications in genome editing and molecular diagnostics.

The determination of biological and chemical factors is important to public health. Herein, we present a metal–organic framework (MOF)-assisted tunable copper hyperprobe (MATCH) for the dynamic on-site detection of diverse targets, including nucleic acids, proteins, antibiotics, and ions. MATCH is synthesized in an ultrafast and mild process by utilizing a MOF to assist mercaptosuccinic acid in capping and reducing Cu²⁺ into a composite hybrid. It exhibits high color-responsiveness to metal ions and hydrogen peroxide. Subsequently, catalase (CAT) is encapsulated within the MOF, forming a CAT@MOF complex, which not only enhances stability and activity but also acts as a signal transducer in MATCH. Through integration with clustered regularly interspaced short palindromic repeats/Cas or immune recognition, the CAT@MOF-mediated MATCH sensor exhibits greater sensitivity and efficiency than gold-standard sensors and remarkably reduces instrument dependence and analysis costs. MATCH provides a multiresponse to H₂O₂, metal ions, nucleic acids, proteins, and antibiotics, which holds great promise for in vitro diagnostics, food safety, and environmental monitoring, thus advancing public health protection.

Point-of-care (POC) sensors have broad use in public security and laboratory medicine. Improving their stability, sensitivity, and quantification analysis is essential but challenging. Herein, we investigate chemical regulations of surface passivation-enhanced emission (sPEE) and aggregation-enhanced emission (AEE) to strengthen the photophysical stability and analytical performance of luminescent copper nanoparticles (CuNPs) for advanced biochemical sensors. Unlike conventional studies focusing simply on enhancing luminescence, we reveal the distinct analytical advantages of different strategies. Surface passivation using poly(acrylic acid) (PAA@CD-CuNPs) and zirconium ion-induced aggregation (Zr⁴⁺@CD-CuNPs) produces over 100-times luminescence enhancement with quantum yield increase from 0.4% to 22% and significantly improves the chemical stability against dissolved oxygen. For H₂O₂ detection, PAA@CD-CuNPs exhibit superior sensitivity, while Zr⁴⁺@CD-CuNPs demonstrate an enlarged linear quantification. sPEE and AEE both effectively strengthen the physicochemical properties of luminescent CuNPs, yet they confer distinct advantages in the relevant analytical performance. These findings provide useful guidance for designing customized POC sensors, particularly when coupled with oxidase- or catalase-catalyzed amplifications (e.g., cholesterol and bacterial detections). For cholesterol detection, PAA@CD-CuNP-based POC sensing shows a broad quantification covering different thresholds (2-11 mM). For Staphylococcus aureus detection, the limit of detection is ∼30 CFU/mL, over 100 times lower than that of reported colorimetric/fluorometric sensors. These assays based on PAA@CD-CuNPs showed 100% accuracy with clinical diagnosis.

Multiplexed and sensitive detection of foodborne pathogens is important and necessary to control foodborne diseases. Mesophilic Clostridium butyricum Argonaute (CbAgo) exhibits endonuclease activity, enabling precise guide DNA (gDNA)-directed target cleavage without sequence constraints, thus holding potential for multiplexed foodborne pathogen detection. The intrinsic activity constraints of CbAgo could be addressed by engineered modification of its gDNA in combination with synergistically enzymatic mechanisms, potentially generating highly efficient cooperative cleavage activity. In this study, the gDNA was engineered to incorporate a DNAzyme-functionalized fragment (designated as cDNA) for substrate strand cleavage, followed by systematic investigation of terminal modification group preferences. The synergistic integration of these dual enzymatic activities ultimately enhanced signal amplification efficacy. Furthermore, we developed a DNAzyme-CbAgo synergistic fluorescence aptamer biosensor, enabling multiplexed detection of three pathogenic bacteria with a detection limit down to 69 CFU mL^-1. This work provides a potential platform for the simultaneous detection of multiplex foodborne pathogenic bacteria without DNA extraction and amplification.