According to data from the World Health Organization, every year, 250，000–500，000 people die from influenza worldwide [1]. The H1N1 virus has high variability and airborne transmission characteristics, posing a serious threat to public health safety. Although vaccination can effectively prevent infection [2], early diagnosis is still a key link in blocking transmission. The current methods for diagnosing influenza A virus include serological testing [3], Virus cultivation [4], Real-time reverse transcription polymerase chain reaction (RT-qPCR) [5], and isothermal amplification techniques (such as Recombinase Polymerase Amplification, Loop-mediated Isothermal Amplification) [6], [7], Among them, RT-qPCR is the gold standard for detecting viruses [8], [9], [10]. However, these methods not only require complex primer design [11]but also require trained professionals and special experimental environments and temperature changes [12], which increases the complexity of operations and diagnostic costs [13], [14], making it difficult to meet the demand for real-time detection in resource-limited areas. Therefore, developing fast and sensitive virus detection methods is considered key to preventing influenza epidemics in the future.

Primer Exchange Reaction (PER) [15], [16], as a novel nucleic acid template extension technique, has shown unique advantages in the field of biosensing in recent years. Its core advantages include: (i) Isothermal amplification characteristics: no need for complex thermal cycling equipment, suitable for on-site detection applications [17]. (ii) Modular design: Different targets can be detected by changing the primer sequence. (iii) Signal amplification capability: It can generate a large number of nucleic acid products with repeated sequences, providing high signal output for downstream detection. Two core mechanisms have been developed for molecular detection strategies based on primer replacement amplification technology: primer-gated mechanism and catalytic hairpin-gated mechanism.

Dynamic DNA nanotechnology enables the construction of modular molecular sensors that can be precisely engineered for direct target recognition and controlled activation of catalytic processes [18]. In the absence of the target nucleic acid, the nanostructured assembly remains intact and enzymatically silent. Upon specific hybridization with the target, the complex undergoes a programmable structural transition that releases potent enzymatic activity, initiating a robust downstream catalytic cascade. A key advantage of this system lies in its customizability, allowing versatile retargeting to diverse disease-specific mutations while enabling efficient signal amplification.

Due to its speed, precision, and high sensitivity, the CRISPR/Cas system has become a leading nucleic acid detection technology in recent years [19], [20], [21]. Currently, the applications of CRISPR/Cas in biosensors for detecting pathogens mainly include Cas12a [22], Cas13a [23], and Cas14a systems [24]. Following activation by either double-stranded DNA (dsDNA) or ssDNA targets, Cas12a acquires two distinct nuclease activities: specific cleavage of target dsDNA and nonspecific trans-cleavage of ssDNA. This dual functionality enables the development of highly sensitive DNA detection biosensors [25]. Cas12a uses a single crRNA to guide the recognition of DNA target sequences, whether in dsDNA or ssDNA configuration. The dsDNA target sequence must have a PAM (5′-TTTN-3′) motif rich in T-nucleotides, while the ssDNA target sequence does not require such a sequence [26]. Therefore, Cas12a trans-cleavage ability has great potential in detecting pathogenic nucleic acids. These technological breakthroughs have laid the foundation for the design of advanced molecular diagnostic tools with exceptional sensitivity. Luan et al. achieved functional conversion from target molecules to primers by constructing catalytic hairpin structures that specifically respond to microRNA (miRNA) [27]. Specifically, the target miRNA interacts with the catalytic hairpin domain, its 3′-hydroxyl end continues to elongate through the catalytic activity of Bst DNA polymerase, causing the appearance of single-stranded DNA products (ssDNA). This can hybridize with fluorescent molecular beacons to restore fluorescence, achieving signal amplification with a detection limit of up to 28.71 fM. Bu et al. were the first to integrate functional DNA aptamers into the locking region of PER catalytic hairpins, constructing a target-responsive molecular switch [28]. Quantitative detection was achieved through electrochemical impedance changes, with a detection limit as low as 19 CFU/mL for Escherichia coli, demonstrating excellent clinical sample detection potential. These two methods achieve target response and signal amplification through differentiated molecular recognition pathways, demonstrating significant innovative value in the field of biosensing. The combination of CRISPR/Cas12a with rolling circle amplification (RCA) was utilized by Zhu and his team in the fabrication of a highly rapid and sensitive biosensor for SARS-CoV-2 [29]. Nevertheless, the application of these approaches is hindered by the intricate composition of pathogen samples and the drawback of low sensitivity.

Inspired by this, we innovatively proposed an enzyme activity-gated PER detection strategy, which is reflected in: (i) The molecular switch design: constructing a DNA polymerase inhibitor complex, releasing enzyme activity inhibition through target-specific binding, and establishing a direct correlation between target recognition and enzyme activity. (ii) Signal transduction mechanism: realizing the three-dimensional signal transduction pathway of enzyme activity → nucleic acid synthesis → CRISPR cleavage. (iii) Pollution prevention and control system: Avoid direct amplification of target nucleic acids and fundamentally eliminate the risk of aerosol pollution. When the target H1N1 virus RNA is present, the target competes with the inhibitor to bind to the antagonist, causing the DNA inhibitor to fall off and the Taq DNA polymerase activity to be restored. Active Taq DNA polymerase catalyzes primer loop extension with the assistance of catalytic hairpin, thereby initiating the PER reaction to generate single-stranded DNA. The trans-cleavage activity of CRISPR/Cas12a is activated by these ssDNA, cutting the HEX/BHQ1 dual-labeled reporter gene and producing a fluorescent signal. On the contrary, when the target H1N1 virus RNA is absent, Taq DNA polymerase remains in a locked state and cannot initiate PER reaction to generate activation substrates, while Cas12a cannot cleave the reporter gene to produce fluorescent signals. This technology integrates the advantages of enzyme activity regulation and CRISPR/Cas12a signal amplification, providing a new solution for molecular diagnostics and has important application value in rapid detection of infectious diseases. Moreover, the presented method demonstrates exceptional sensitivity and high programmability in detecting RNA viruses, attributes that are critically important for enabling early diagnosis, facilitating effective intervention strategies, and controlling the spread of infectious diseases.