Meat is a critical component of the human diet and supplies essential nutrients such as proteins, fatty acids, and vitamins. However, owing to their high protein content and water activity, meat products are susceptible to deterioration during processing, transportation, and storage, leading to food safety concerns and resource waste. Currently, the total viable count (TVC) serves as an important indicator in food microbiology testing for assessing meat freshness. Nevertheless, the traditional TVC plate count method has several limitations, such as complex operation, a lengthy determination time, and sensitivity to environmental conditions. Adenosine triphosphate (ATP) is recognized as the “small-molecule currency” of intracellular energy transfer and plays a pivotal role in regulating cellular metabolism (Li, Peng, et al., 2024). In addition to being closely associated with a range of diseases, such as inflammation, sepsis, and malignancy in mammals (Li, Wang, et al., 2024), abnormal ATP levels also indicate the extent of microbial contamination in food (Liu et al., 2019; Spari & Beldi, 2020). Numerous studies have demonstrated that ATP content is strongly correlated with meat spoilage, making it a primary indicator for evaluating freshness in clinical microbiology and food quality control (Oshita et al., 2011; Oto et al., 2013; Su et al., 2024). Furthermore, according to the national standard of the People's Republic of China (GB/T 36004–2018), the ATP bioluminescence method is employed to quantify ATP levels on food-contact surfaces (e.g., packaging materials, containers, and tableware) to assess residual biomass. Therefore, the development of portable, precise, and ultrasensitive ATP detection platforms is crucial for ensuring food quality, advancing clinical diagnosis, and monitoring industrial hygiene.

To date, various sensing strategies based on fluorescence (Peng et al., 2020), colorimetry (Li et al., 2019), electrochemistry (Zhang et al., 2018), chemiluminescence (Song et al., 2014), and surface-enhanced Raman spectroscopy (SERS) (Shi et al., 2017) have been extensively employed for ATP detection. All these reported methods use classical DNA aptamers for adenosine and ATP as analyte-specific recognition elements (Lin & Patei, 1997). However, the dissociation constant (Kd) between the DNA aptamer with this sequence and ATP is relatively high (Kd ∼ 6 μM) (Ding & Liu, 2023), and the fact that the recognition sites are adenine and ribose rather than phosphate groups limits the selection of ATP and its analogs, such as adenosine monophosphate, adenosine diphosphate, and adenosine (Sassanfar & Szostak, 1993). This has resulted in the suboptimal sensitivity and specificity of these methods. Enzymes exhibit remarkable substrate specificity, and among them, T4 DNA ligases demonstrate a specific dependence on the cofactor ATP. Activated T4 DNA ligase efficiently catalyzes phosphodiester bond formation between adjacent 5′-phosphate and 3′-hydroxyl termini within duplex DNA or RNA, thereby providing an effective platform for constructing a highly selective detection system for ATP (Shuman, 2009). Furthermore, the synergistic integration of various amplification techniques to minimize background signals and maximize the desired signal is a widely adopted strategy aimed at improving detection sensitivity in sensing approaches (Cheng et al., 2022).

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) systems are nuclear endonuclease effectors of the adaptive immune system of archaea and bacteria (Jinek et al., 2012). These systems serve as a defense mechanism against invading foreign genetic material, such as viruses, and have emerged as revolutionary and effective tools in genome editing (Cong et al., 2013; Mali et al., 2013). Owing to their impressive nuclease cleavage ability, which is regarded as “God's scissors”, these systems have also triggered the innovation of in vitro molecular diagnostic technology, such as the CRISPR–Cas12a (Cpf1)-based detection platform DNA endonuclease-Targeted CRISPR Trans Reporter (DETECTR) (Chen et al., 2018). In the CRISPR–Cas12a system, the guide CRISPR RNA (crRNA) initially binds to Cas12a. It then specifically recognizes the target DNA as an activator, including single-stranded DNA (ssDNA) or specific double-stranded DNA (dsDNA) containing a T nucleotide-rich protospacer-adjacent motif (PAM), to form a ternary complex. Activated Cas12a exhibited deoxyribonuclease (DNase) activity, which not only cleaves target DNA (cis-cleavage) but also indiscriminately cleaves nearby nontarget ssDNA substrates (trans-cleavage) (Li, Cheng, Liu, et al., 2018). The One-HOur Low-cost Multipurpose Highly Efficient System (HOLMES), a nucleic acid diagnostic tool, integrates the CRISPR–Cas12a system with polymerase chain reaction (PCR) to increase detection sensitivity and target specificity (Li, Cheng, Wang, et al., 2018). In contrast to PCR, which requires specialized thermal cycling equipment, isothermal amplification techniques such as hybridization chain reaction (HCR) (Ma et al., 2023), recombinase polymerase amplification (RPA) (Xu et al., 2022), loop-mediated isothermal amplification (LAMP) (Zhang et al., 2021), and rolling circle amplification (RCA) are commonly chosen for biosensing (Qing et al., 2021). Among them, RCA has been favored by researchers because of its advantages of simple operation, wide application range and efficient in vitro exponential amplification ability (Yue et al., 2021). More importantly, the activation of the RCA reaction depends on the hybridization of primers with complementary padlock probes, and it generates special long-stranded repetitive DNA that can be used as an activator of CRISPR–Cas12a, which not only improves the specificity of the analysis but also creates the possibility of combining CRISPR–Cas12a with cofactor-dependent enzymatic reactions.

Currently, biosensors based on the CRISPR–Cas12a system are booming. Most of these methods rely on a single optical sensing strategy that involves fluorescence quenching between a fluorophore and a quencher, as well as fluorescence signal recovery during Cas12a trans-cleavage of ssDNA (Zhu et al., 2023). However, the bulky and expensive nature of signal readout devices limits their use in nonlaboratory and resource-poor settings. To address this issue, using user-friendly portable devices that provide fast readings is crucial for expanding biosensor application scenarios. Point-of-care (POC) diagnostic devices are indispensable to the modern public healthcare system, enabling real-time delivery of precise diagnostic data to clinicians in resource-limited or remote settings, thereby facilitating informed therapeutic interventions (Syedmoradi et al., 2017). Small hand-held devices, such as glucometers, are highly favored due to their “pocket” size, user friendliness, and cost effectiveness (He et al., 2023). In July 2011, Yu Xiang and Yi Lu published an innovative article demonstrating the quantification of nonglucose targets via DNA functionalization of invertases with glucometers (Xiang & Lu, 2011).

Herein, we present a novel strategy integrating CRISPR–Cas12a with a portable glucometer for the sensitive detection of ATP. This strategy employs cofactor-dependent enzymatic ligation-triggered RCA, thereby enabling signal conversion and amplification of the target ATP. As illustrated in Fig. 1, the process begins with the prehybridization of primers and padlock probes via Watson–Crick base pairing. The presence of the target ATP activates ATP-dependent T4 DNA ligase, leading to the circularization of the padlock probe. The circularized padlock probe initiates RCA, resulting in the generation of numerous DNA amplicons with repetitive fragments (initiator DNA), thereby activating the trans-cleavage activity of CRISPR-Cas12a to cleave ssDNA and release the invertase modified on the surface of the magnetic beads. The released invertase catalyzes the hydrolysis of sucrose into glucose, enhancing the glucometer signal. The signal intensity of the glucometer is directly proportional to the concentration changes of the target ATP. By measuring the ATP level on the surface of meat and correlating it with TVC, the freshness of meat can be effectively assessed. Leveraging the specific dependence of the enzyme ligation reaction on ATP as a cofactor, this strategy facilitates the construction of a highly selective ATP-targeted biosensing platform. Moreover, two consecutive signal amplification steps (a target-responsive RCA reaction and a CRISPR–Cas12a-catalyzed trans-cleavage reaction) enable high-sensitivity detection of ATP, with a limit of detection (LOD) of 1.08 nmol/L. Similarly, the ssDNA-invertase-modified magnetic bead (I-M probe) was switched to a classical ssDNA fluorescent reporter probe (F-Q probe) composed of carboxyfluorescein (FAM) and black hole quencher 1 (BHQ1) pairs at both termini, which were cleaved to enhance the fluorescence signal for verifying the accuracy of this strategy.