Catalytic Hairpin Assembly-Aided CRISPR/Cas-SERS for Nucleic Acid Detection Enhanced by Multiple Catalytic Amplification

An integrated signal amplification strategy based on catalytic hairpin assembly and hybridization chain reaction for driving a CRISPR/Cas12a biosensor toward ultrasensitive detection of microRNAs

HCR-assisted CRISPR/Cas12a platform enables logic-gate and multimodal detection of kanamycin and bisphenol A.

A novel triple-modal biosensor integrating hybridization chain reaction (HCR) with CRISPR/Cas12a was developed for sensitive and selective detection of kanamycin (KANA) and bisphenol A (BPA). The system employs a phosphorothioate-modified G-rich hairpin (SHG4-2) as a dual-functional reporter probe, which resists Cas12a trans-cleavage and enables multimodal signal output via SG-quadruplex (SG4) formation. Upon target recognition by aptamers, an initiator strand is released to trigger HCR amplification, generating long double-stranded DNA products that activate Cas12a trans-cleavage. This cleaves the (SHG4-2) probe, releasing SG-rich sequences that self-assemble into SG4 structures, yielding fluorescence (with Thioflavin T), colorimetric (via SG4/hemin-catalyzed TMB oxidation), and smartphone-readable RGB signals. This platform enables parallel detection of a single target analyte, allowing flexible detection modes. Under optimized conditions, the sensor achieved detection limits as low as 20.1 pM for KANA and 6.8 pM for BPA in fluorescence mode, 32.3 pM for KANA and 17.1 pM for BPA in colorimetric mode, and 74.3 pM for KANA and 35.8 pM for BPA in smartphone mode, with excellent selectivity against interfering analogues. Successful application in spiked milk samples demonstrated high recovery rates and good reproducibility. In addition, the platform supports the logical gate operations of OR (single-target detection) and AND (dual-target detection), which allows flexible detection modes. This work presents a versatile, amplification-enhanced multimodal sensing strategy for environmental and food safety monitoring, highlighting its potential for logic-driven biosensing applications.

A Novel CRISPR/Cas13a sensor for highly sensitive detection of DENV based on AuPt/g-C3N4@GO and Ag@NU-1000.

Dengue virus, an arthropod-borne pathogen, poses a significant global public health concern, necessitating the development of sensitive and rapid diagnostic assays. This paper introduces a novel biosensing platform designed for the ultrasensitive detection of dengue viral RNA. The platform integrates recombinase polymerase amplification, CRISPR/Cas13a-based recognition, and electrochemiluminescence resonance energy transfer (ECL-RET). The sensor employs a highly efficient luminescent emitter, which is a ternary composite of gold-platinum nanoparticles supported on graphitic carbon nitride and graphene oxide. A suitable energy acceptor, consisting of silver-decorated metal-organic frameworks, effectively quenches the emitter's signal via resonance energy transfer. Upon the introduction of target nucleic acids, RPA rapidly generates numerous amplicons. These amplicons are specifically recognized by the CRISPR/Cas13a system, which subsequently activates the collateral cleavage activity of Cas13a. This enzymatic activity cleaves a DNA linker that tethers the acceptor to the electrode surface, leading to the release of the acceptor and a subsequent restoration of the electrochemiluminescence intensity. The recovered signal exhibits a linear proportionality to the target concentration across a broad dynamic range, spanning from 0.1 fg/mL to 100 ng/mL. The detection limits for the four dengue serotypes range from 1.09 to 2.5 fg/mL. The assay demonstrates excellent specificity, showing no cross-reactivity with Zika, Japanese encephalitis, or West Nile viruses, and also exhibits good reproducibility. By combining isothermal amplification, specific CRISPR targeting, and a sensitive luminescence readout, this work presents a straightforward and robust method for the early diagnosis of dengue and for outbreak surveillance. Furthermore, this platform can be readily adapted for the detection of other pathogens of interest.

Programmable Allosteric DNAzyme Coupled with CRISPR/Cas12a System for Multiplexed and Sensitive Detection of Extracellular Vesicle Derived MicroRNAs

An electrochemiluminescence biosensor for sulfadimethoxine detection based on enzymatic DNA walker-activated CRISPR/Cas12a cascade for signal enhancement.

Sulfadimethoxine (SDM) is a widely used veterinary antibiotic. Its residues in food and the environment may cause bacterial resistance and threaten human health. In this study, a novel electrochemiluminescence (ECL) biosensor with signal amplification was developed for the sensitive detection of SDM. A g-C3N4@Au composite was modified on the electrode surface, generating a strong ECL signal with K2S2O8 as the coreactant. Hairpin DNA terminated with ferrocene carboxylic acid (FcA) was self-assembled onto g-C3N4@Au via thiol-gold bonds, causing Fc to quench the electrochemiluminescence signal of g-C3N4. Upon SDM addition, it specifically bound to the aptamer strand, releasing the DNA Walker. The DNA walker then continuously hybridized with the activator strand S1 and was cleaved by the nicking endonuclease Nt.BsmAI, generating a large number of S1. S1 further activated the trans-cleavage activity of CRISPR/Cas12a, which cleaves the hairpin DNA on the electrode, releasing Fc and recovering the ECL signal. By monitoring the ECL intensity change during the "signal-off" to "signal-on" transition, quantitative detection of SDM was indirectly realized. The detection range for SDM is 1.0 × 10-14 to 1.0 × 10-7 mol L-1, with a detection limit of 7.15 × 10-15 mol L-1. The sensor was successfully applied to the detection of SDM in food and water samples. Benefiting from the catalytic cascade amplification involving multiple enzymes, the developed method demonstrated high ultrahigh sensitivity.

A low-cost CHA-integrated CRISPR/Cas12a-based test strip platform for on-site gene detection.

On-site nucleic acid detection plays a crucial role in disease diagnosis, biosafety monitoring, and food quality control. This study develops a novel nucleic acid detection platform that integrates catalytic hairpin assembly (CHA) with the CRISPR/Cas12a system and utilizes pregnancy test strips (PTS) for result visualization, addressing the limitations of existing nucleic acid detection methods in balancing sensitivity, specificity, and portability with cost and dependence on a cleanroom. The main mechanism involves the following three steps. The presence of target RNA triggers the CHA reaction, generating double-stranded DNA (dsDNA) as an activation unit. Subsequently, this unit activates the CRISPR/Cas12a system to specifically cleave the single-stranded DNA (ssDNA) that has bridged human chorionic gonadotropin (HCG) to a magnetic bead, ultimately releasing HCG that produces a visual result on the PTS. This dual-signal amplification strategy (CHA cycling and Cas12a trans-cleavage) can detect concentrations as low as 10 pM in approximately 50 min, without the need for pre-amplification of the target nucleic acid. This detection system ensures high sensitivity and specificity while effectively avoiding non-specific activation. In practical applications with transgenic maize samples, the detection results are highly consistent with those of real-time quantitative polymerase chain reaction (qPCR), validating its reliability in real-world scenarios. This innovative method offers advantages such as simple operation and low cost, providing an efficient tool for rapid nucleic acid detection while demonstrating broad potential for application in resource-limited settings.