Leucine aminopeptidase (LAP), a kind of proteolytic enzyme and an indicator of many kinds of cancer. Developing effective methods for tracking endogenous LAP activity is crucial for LAP-related cancer diagnosis and treatment. In this work, we report a new near-infrared fluorescent probe: CY-P, which is capable of highly effective monitoring of endogenous LAP activity in vitro and in vivo. CY-P was constructed by incorporating a dipeptide (Cys-Leu: Cysteine-Leucine) into a water-soluble NIR-emitting fluorophore (CY–OH: a semi-cyanine scaffold). Upon exposure to LAP, the amide bond in Cys-Leu will be specifically cleaved by LAP, and subsequent an intramolecular cyclization to release the unmasked phenol-based semi-cyanine. In this process, a dramatic fluorescence enhancement at 701 nm was observed, the evaluation of LAP activity is established in terms of the relationship between fluorescence light-up efficiency and LAP activity. In our experiments, CY-P features high feedback towards LAP with various advantages, such as low detection limit (4.9 × 10⁻⁵ U mL⁻¹), fast-response (∼9 min), good biocompatibility, and NIR emission. As expected, the probe exhibits low cytotoxicity and excellent cell membrane permeability, which is successfully used to monitor endogenous LAP activity in living cancer cells and zebrafish models. Therefore, we anticipate that this newly designed fast LAP detection platform will provide an alternative method for the studies of related diseases.

亮氨酸氨肽酶（LAP），一种蛋白水解酶，是多种癌症的指标。开发跟踪内源性 LAP 活动的有效方法对于 LAP 相关癌症的诊断和治疗至关重要。在这项工作中，我们报告了一种新的近红外荧光探针：CY-P，它能够在体外和体内高效监测内源性 LAP 活性. CY-P 是通过将二肽（Cys-Leu：半胱氨酸-亮氨酸）掺入水溶性 NIR 发射荧光团（CY-OH：半花青支架）来构建的。暴露于 LAP 后，Cys-Leu 中的酰胺键将被 LAP 特异性裂解，随后进行分子内环化以释放未掩蔽的酚基半花青。在此过程中，观察到 701 nm 处的荧光显着增强，根据荧光点亮效率和 LAP 活性之间的关系建立了 LAP 活性的评价。在我们的实验中，CY-P 具有对 LAP 的高反馈，具有各种优势，例如检测限低（4.9 × 10 ^-5 U mL ^-1)、快速响应（~9 分钟）、良好的生物相容性和 NIR 发射。正如预期的那样，该探针具有低细胞毒性和优异的细胞膜通透性，已成功用于监测活癌细胞和斑马鱼模型中的内源性 LAP 活性。因此，我们预计这种新设计的快速 LAP 检测平台将为相关疾病的研究提供一种替代方法。