Forster Resonance Energy Transfer-Based Self-Assembled Nanoprobe for Rapid and Sensitive Detection of Postoperative Pancreatic Fistula
Forster Resonance Energy Transfer-Based Self-Assembled Nanoprobe for Rapid and Sensitive Detection of Postoperative Pancreatic Fistula
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DOI：
10.1021/acsami.5b11902
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发表时间：
2016-03-02
影响因子：
9.5
通讯作者：
Hashizume, Makoto
中科院分区：
文献类型：
--
作者：
Hamano, Nobuhito;Murata, Masaharu;Hashizume, Makoto
术后胰瘘(POPF)是胃肠外科手术后最严重、最具挑战性的并发症。从器官残留物中渗出的活化的胰液含有攻击周围组织的蛋白酶，可能会导致严重的炎症、组织坏死和瘘管的形成。然而，很难在手术中观察胰腺渗漏，也很难评估患者床边渗漏液体的蛋白水解酶活性。本报告描述了一种基于蛋白质纳米笼的蛋白质酶比率传感器，它包括一个胰腺蛋白酶敏感的小热休克蛋白(HSP)16.5，它是一种自然存在的蛋白，通过24个亚基的自组装形成球形结构，以及一个化学连接的供体受体染料对，用于Forster共振能量转移(FRET)。HSP-FRET探针是通过每个染料标记的工程热休克蛋白的亚基交换构建的，得到了直径约10 nm的球形纳米笼，对血浆中的降解表现出非常高的稳定性，并且对小鼠没有明显的毒性。FRET的效率取决于染料的取向和受体/供体的比例。利用HSP-FRET纳米探针，用高特异性的荧光回收法定量分析了胰腺蛋白水解酶，包括胰蛋白酶、α-胰凝乳蛋白酶和弹性蛋白酶。此外，HSP-FRET纳米探针具有足够的灵敏度，仅用肉眼就能在10分钟内检测到患者胰液中的POPF。因此，这种新型的纳米探针被认为是胃肠外科手术中检测POPF和显示激活的胰液的一种有效和方便的工具。
Postoperative pancreatic fistula (POPF) is the most serious and challenging complication following gastro-enterological surgery. Activated pancreatic juice leaking from the organ remnant contains proteases that attack the surrounding tissue, potentially leading to severe inflammation, tissue necrosis, and fistula formation. However, it is difficult to observe pancreatic leakage during surgery and to evaluate the protease activity of leaked fluid at the patient's bedside. This report describes a protein nanocage-based protease ratiometric sensor comprising a pancreatic protease-sensitive small heat-shock protein (HSP) 16.5, which is a naturally occurring protein in Methanococcus jannaschii that forms a spherical structure by self-assembly of 24 subunits, and a chemically conjugated donor acceptor dye pair for Forster resonance energy transfer (FRET). The HSP-FRET probe was constructed by subunit exchange of each dye-labeled engineered HSP, resulting in a spherical nanocage of approximately 10 nm in diameter, which exhibited very high stability against degradation in blood plasma and no remarkable toxicity in mice. The efficiency of FRET was found to depend on both the dye orientation and the acceptor/donor ratio. Pancreatic proteases, including trypsin, a-chymotrypsin, and elastase, were quantitatively analyzed by fluorescence recovery with high specificity using the HSP-FRET nanoprobe. Furthermore, the HSP-FRET nanoprobe was sufficiently sensitive to detect POPF in the pancreatic juice of patients using only the naked eye within 10 min. Thus, this novel nanoprobe is proposed as an effective and convenient tool for the detection of POPF and the visualization of activated pancreatic juice during gastroenterological surgery.