Adenosine triphosphate (ATP), the main carrier of chemical energy, plays a key role in various biochemical reactions such as cellular metabolism. Currently, ATP levels are considered important indicators of microbial content in food safety, and food freshness can be determined by detecting ATP content. Some ATP sensing strategies have been applied to evaluate food freshness. However, cumbersome nanomaterial preparation, low sensitivity, and low reliability hamper their widespread application. Herein, a simple, high-performance, and reliable dual-mode sensing system based on hemin-G-quadruplex (G4) DNAzyme was established to detect ATP and assess fish freshness. Two nucleic acid probes, including subunits of the hemin-G4 DNAzyme in inactive structures and anti-ATP aptamer, self-assemble upon the input of ATP into the active hemin-G4 DNAzyme unit. The generated DNAzyme acts as a biocatalyst for colorimetric or fluorescent readout of the sensing process. The colorimetric and fluorescent dual-mode sensing system enables highly sensitive and reliable analysis of target ATP with detection limits of 71 nM and 73 nM, respectively. Moreover, the biosensor exhibited good selectivity for differentiating ATP from other interfering analytes. The proposed system was used to detect ATP in perch samples, and a linear correlation between ATP level and microbial content was confirmed. The established ATP-sensing system reliably evaluated fish freshness. Notably, in comparison with microbiological counts, the proposed DNAzyme-based dual-mode strategy for freshness evaluation is facile, highly efficient, and cost-effective, thus providing a promising method for food safety and quality monitoring.

三磷酸腺苷（ATP）是化学能的主要载体，在细胞代谢等多种生化反应中起着关键作用。目前，ATP水平被认为是食品安全中微生物含量的重要指标，通过检测ATP含量可以判断食品的新鲜度。一些 ATP 传感策略已应用于评估食物新鲜度。然而，繁琐的纳米材料制备、低灵敏度和低可靠性阻碍了它们的广泛应用。在此，建立了一种基于血红素-G-四链体 (G4) DNAzyme 的简单、高性能和可靠的双模式传感系统来检测 ATP 和评估鱼类新鲜度。两个核酸探针，包括无活性结构中的血红素-G4 DNAzyme 亚基和抗 ATP 适体，在 ATP 输入到活性血红素-G4 DNAzyme 单元时自组装。生成的 DNAzyme 充当生物催化剂，用于传感过程的比色或荧光读出。比色和荧光双模式传感系统能够对目标 ATP 进行高度灵敏和可靠的分析，检测限分别为 71 nM 和 73 nM。此外，该生物传感器在区分 ATP 和其他干扰分析物方面表现出良好的选择性。该系统用于检测鲈鱼样本中的 ATP，证实了 ATP 水平与微生物含量之间的线性相关性。已建立的 ATP 传感系统可以可靠地评估鱼的新鲜度。值得注意的是，与微生物计数相比，所提出的基于 DNAzyme 的双模式新鲜度评估策略简便、高效且具有成本效益，从而为食品安全和质量监测提供了一种有前途的方法。