Protease assay by activated fluorescent self-assembled gold nanoparticles applied in pancreatitis diagnosis
|關鍵字:||急性胰臟炎;生物感測;胰凝乳蛋白酶;奈米金粒子;胜肽;acute pancreatitis;biosensor;chymotrypsin;gold nanoparticles;peptide|
|摘要:||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。
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.