Hazards Monitoring Bionano Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, South Korea; Department of Polymer Engineering, Graduate School, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 61186, South Korea.
Hazards Monitoring Bionano Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, South Korea; University of Science & Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, South Korea.
Biosens Bioelectron. 2018 May 15;105:151-158. doi: 10.1016/j.bios.2018.01.018. Epub 2018 Jan 10.
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 1.0UmL with a 10-10UmL limit of detection (LOD), showing a response rate completed within 1.5min. 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）是成骨细胞早期分化过程中成骨细胞活性的关键生物标志物，但可用于检测其的生物兼容方法很少。在这里，我们描述了用于 ALP 荧光检测的高灵敏度和快速响应新型近红外（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，并显著改变其光物理性质，因此产生了来自于荧光部分的催化水解的大的“开启”荧光信号。我们的测定法可定量检测 0 至 1.0UmL 的 ALP 活性，检测限（LOD）为 10-10UmL，显示出在 1.5 分钟内完成的响应速度。这是一种在生物系统中探测 ALP 活性的潜在强大方法，可通过实时监测活细胞和动物内源性 ALP 的浓度和时间依赖性变化来实现。基于磷酸部分与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）支架可进行骨样支架内的 ALP 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，有可能在骨缺损内新骨形成的早期阶段检测到 ALP。
