Alkaline phosphatase (ALP) is a critical biological marker for osteoblast activity during early osteoblast differentiation, but few biologically compatible methods are available for its detection. Here, we describe the discovery of highly sensitive and rapidly responsive novel near-infrared (NIR) fluorescent probes (NIR-Phos-1, NIR-Phos-2) for the fluorescent detection of ALP. ALP cleaves the phosphate group from the NIR skeleton and substantially alters its photophysical properties, therefore generating a large “turn-on” fluorescent signal resulted from the catalytic hydrolysis on fluorogenic moiety. Our assay quantified ALP activity from 0 to 1.0 U mL⁻¹ with a 10⁻⁵−10⁻³ U mL⁻¹ limit of detection (LOD), showing a response rate completed within 1.5 min. A potentially powerful approach to probe ALP activity in biological systems demonstrated real-time monitoring using both concentration- and time-dependent variations of endogenous ALP in live cells and animals. Based on high binding affinity to bone tissue of phosphate moiety, bone-like scaffold-based ALP detection in vivo was accessed using NIR probe-labeled three-dimensional (3D) calcium deficient hydroxyapatite (CDHA) scaffolds. They were subcutaneously implanted into mice and monitored ALP signal changes using a confocal imaging system. Our results suggest the possibility of early-stage ALP detection during neo-bone formation inside a bone defect, by in vivo fluorescent evaluation using 3D CDHA scaffolds.

碱性磷酸酶（ALP）是早期成骨细胞分化过程中成骨细胞活性的关键生物学标志物，但几乎没有生物学兼容的方法可用于其检测。在这里，我们描述了用于ALP荧光检测的高度敏感和快速响应的新型近红外（NIR）荧光探针（NIR-Phos-1，NIR-Phos-2）的发现。ALP从NIR骨架上切割磷酸基团，并实质上改变了其光物理性质，因此产生了大的“开启”荧光信号，这是由荧光部分的催化水解产生的。我们的测定法定量了从0到1.0 U mL ^-1的ALP活性，而10 ^-5 -10 ^-3  U mL ^-1检测限（LOD），显示在1.5分钟内完成的响应速度。在生物系统中探测ALP活性的一种潜在强大方法证明了使用活细胞和动物体内内源性ALP的浓度和时间依赖性变化实时监测。基于与磷酸盐部分对骨组织的高结合亲和力，使用NIR探针标记的三维（3D）缺钙羟基磷灰石（CDHA）支架可进行基于骨样支架的ALP体内检测。将它们皮下植入小鼠体内，并使用共聚焦成像系统监测ALP信号变化。我们的结果表明，通过使用3D CDHA支架进行体内荧光评估，可以在骨缺损内部新骨形成期间进行早期ALP检测。