酵素活化自組裝式螢光奈米金粒子檢測平台之建立並應用於胰臟炎的診斷

Abstract

奈米粒子目前廣泛被應用於生物感測，其中尤以奈米金粒子(AuNPs)最常被應用，此乃AuNPs具有易於被生物分子修飾和共振能量轉移的特性。本研究所設計的生物檢測原理係以AuNPs具有遮蔽螢光之特性，其擁有與距離有關的廣效波長遮蔽螢光特性，據此我們建立了一個快速檢測蛋白酵素(proteinase)活性的平台，並探討胜肽受質的序列設計，用以獲得較高靈敏度的檢測。我們利用螢光基團FITC標記的胜肽受質結合上AuNPs，此為一活化自組裝式螢光AuNPs探針，其被應用於蛋白酵素活性檢測中，原理是當酵素水解胜肽受質時，其尾端的螢光基團可遠離AuNPs表面，使其螢光波長得以被偵測。本研究首先以具有高活性的蛋白酶K (proteinase K)為檢測對象，並藉以建立最佳化檢測條件和受質胜肽序列設計原則。接著我們將上述平台改裝用以胰凝乳蛋白酶(chymotrypsin)的活性檢測，並運用於胰臟功能的檢測。 為了要增加螢光AuNPs探針對酵素的靈敏性，本研究共設計三條胜肽基質用於製作AuNPs探針，其設計著重於降低AuNPs表面的立體障礙以利蛋白酵素辨識，並藉由延伸胜肽的長度或改變特定序列以有效地增加AuNPs探針偵測酵素活性的能力。實驗結果顯示，GPLGLARGGGGGC之AuNPs探針用於偵測蛋白酶K與胰凝乳蛋白酶活性，其較GPLGLAG(Hyp)C之AuNPs探針用於檢測分別提升了3和10倍螢光強度的變化；而利用GPLGLARDDDDDC之AuNPs探針用於酵素活性的檢測，其偵測極限可以從ng/mL下降到pg/mL程度，且偵測時間只需15分鐘。以上結果證明受質胜肽序列的設計對螢光AuNPs探針應用於檢測蛋白酵素活性上極為重要。 AuNPs探針GPLGLARDDDDDC進一步被應用於生物樣本的檢測，包括腸液與糞便檢體中胰凝乳蛋白酶活性的檢測，而在實驗小鼠模式中，單顆小鼠糞便即可簡易地利用我們所設計的AuNPs探針來測定其胰凝乳蛋白酶活性。本研究探討胰凝乳蛋白酶在小腸中的活性分佈，此實驗為比較在禁食與進食控制下，小鼠小腸液中的胰凝乳蛋白酶活性變化，結果顯示在不同飲食控制下空腸與迴腸中胰凝乳蛋白酶活性會有顯著的差異，而其糞便中胰凝乳蛋白酶活性變化也可對應其禁食與進食狀況。本研究也建立利用蛙皮素(cerulein)腹腔注射誘發急性胰臟炎的小鼠模式，再利用所設計之AuNPs探針偵測其小腸與糞便中胰凝乳蛋白酶活性變化，期可作為評估急性胰臟炎的檢測指標。實驗結果顯示在急性胰臟炎小鼠的十二指腸腸液與糞便中，其胰凝乳蛋白酶活性分別顯著下降到正常對照組的25%與30%。 本研究所建立的酵素活化自組裝式螢光AuNPs檢測平台有潛力成為高靈敏且可快速偵測的生物檢測平台，也被證實具有臨床應用的可行性。而所建構的AuNPs探針可藉由受質胜肽序列的置換，即可進一步應用於其他酵素活性的檢測，此使本研究之成果更具可利用性與價值性。

Nanoparticles are usually used in biosensing field and among all of the nanoparticles, gold nanoparticles (AuNPs) are most widely applied. It is because that AuNPs are easy to be conducted with surface biomolecule modification and possess the characteristic of resonance energy transfer. The principle of biosensing platform designed in this study is based on AuNPs provide quenching fluorescence ability and which is wide range wavelength quenching and distance dependence. According to the property, a rapid proteases activity sensing platform was established and the peptide substrates design in order to gain high sensitivity detection platform was also investigated. FITC labeled peptide substrates were used to conjugate onto AuNPs to be an activated fluorescent self-assembled AuNPs (AuNPs probe) that are used for the activity assay of proteases. The detecting mechanism is that proteases could cleavage peptide substrates and then the fluorephore at the end of peptide substrates could diffuse away from AuNPs surface, which the fluorephore emitting wavelength could be detected. In this study, proteinase K with pretty high specific activity was first used as target proteases to establish optimal detecting conditions and to evaluate the design principle of peptide substrates. After that, the established platforms were used in chymotrypsin activity assay and applied in the estimation of pancreatic function. For increasing the sensitivity of proteases to AuNPs probe, there were three peptides substrates, i.e. AuNPs probes, designed and evaluated in the present study. The design was emphasized on the decreasing steric barrier on AuNPs surface by extending length of peptide or changing specific sequences of AuNPs to increase sensitivity of the AuNPs probes used in the assay of proteases activity. The results indicate that the AuNPs probe with GPLGLARGGGGGC could increase the detecting sensitivity of proteinase K and chymotrypsin by approximate 3 and 10 folds fluorescent intensity change compared with those by the AuNPs probe of GPLGLAG(Hyp)C, respectively. Moreover, the AuNPs probe with GPLGLARDDDDDC applied in proteases activity assay could lower the detection limit from ng/mL to pg/mL level and the detection time was only 15 min. The results above indicate that the design of peptide sequence plays important role in the AuNPs probe applied in protease assay. The AuNPs probe with GPLGLARDDDDDC was further applied in the detection of biological samples, including the activity assay of intestinal and fecal chymotrypsin. For the experimental mouse model, simply one feces is needed for chymotrypsin activity assay by the AuNPs probe developed in this study. The chymotrypsin activity distributions in intestinal fluids of mice were investigated by a procedure of fasting/feeding control. The results show a significant difference in jejunum and ileum under diet controls; besides, the activity change of fecal chymotrypsin also corresponds to different situations of fasting and feeding. Acute pancreatitis (AP) mouse model induced by intraperitoneal injection of cerulein was established in this study. The AuNPs probe was applied to evaluate chymotrypsin activity level in intestine and feces with the expectation of being an indicator of AP. The results indicate that chymotrypsin in duodenal fluid and feces significantly decrease and remain only 25% and 30 % activity level compared with those in normal subjects, respectively. The activated fluorescent self-assembled AuNPs probe established in this study shows the potential to be a biosensing platform with high sensitivity and rapid detection, and has been approved to be available in clinical applications. For wide development by simply replacing the efficient peptide substrates for different protease targets can be rationally expected, it makes the achievements procured this study being more profitably and valuably.