以解離放光染料及分子轉子為基底之螢光增益探針應用於蛋白質偵測

Application of Disassembly-Emission Dye and Molecular Rotor in Fluorescence Turn-on Detection of Proteins

吳盈漪(Wu, Ying Yi)

指導教授 ： 陳貴通

國立清華大學/理學院/化學系/碩士(2015年)

摘要

小分子螢光探針具有高選擇性、高靈敏度且可快速偵測等優點。因此，近年來已有許多用來偵測酶蛋白的螢光探針，藉由酶的催化反應使非螢光基團的受質結構改變造成螢光訊號的上升。然而此方法並不適用於偵測非酶蛋白，使得非酶蛋白的螢光探針在設計上仍有許多限制。在此我們設計了一種可對非酶蛋白具專一性偵測之近紅外光螢光探針，此探針是由Cy5螢光基團修飾上小分子配體而組成，其螢光增益機制是透過探針自組裝形成J型聚集而淬熄螢光。加入目標分析物後，親和性配體會和目標分析物結合使聚集後的探針回復成單體，在近紅外光波段可觀察到顯著的螢光增益。我們將此概念應用於人類碳酸酐酶之專一性檢測，並成功標記細胞表面之癌症標記物：跨模型碳酸酐酶IX，且不需多餘的清洗步驟。
此外，我們也設計了另一個以分子轉子為基底的螢光探針，此種螢光分子在水溶液中，單鍵可自由旋轉造成非放光性的能量損失，加入目標蛋白後，藉由目標蛋白和探針結合後抑制探針的單鍵旋轉，使得螢光訊號顯著上升。我們成功將此設計應用於人類血清白蛋白之偵測上，人類血清白蛋白可做為腎臟疾病及非糖尿病患者心血管疾病的檢測指標。加入人類血清白蛋白後，此探針的螢光增益倍數高達400倍之多，且具有高選擇性和高靈敏度，我們也成功的將此探針應用於尿液中白蛋白之定量分析上。

關鍵字

螢光探針 ； 細胞顯影 ； 分子轉子

並列摘要

Currently most of the fluorogenic probes are designed for the detection of enzymes which work by converting the non-fluorescence substrate into the fluorescence product via an enzymatic reaction. On the other hand, the design of fluorogenic probes for non-enzymatic proteins remains a great challenge. Herein, we report a general strategy to create near-IR fluorogenic probes, where a small molecule ligand is conjugated to a novel γ-phenyl-substituted Cy5 fluorophore, for the selective detection of proteins through a non-enzymatic process. Detail mechanistic studies reveal that the probes self-assemble to form fluorescence-quenched J-type aggregate. In the presence of target analyte, bright fluorescence in the near-IR region is emitted through the recognition-induced disassembly of the probe aggregate. Based on this design, a fluorogenic probe for hCA II detection was constructed and was applied for the no-wash imaging of tumor cells for the detection of hypoxia-induced cancer-specific biomarker, transmembrane-type carbonic anhydrase IX.
We have also created another fluorogenic probe which belongs to a class of fluorescent dyes called molecular rotors for the detection of human serum albumin (HSA), a key indicator for the early diagnosis of renal disease and for the cardiovascular disease in non-diabetic individuals. In aqueous buffer, molecular rotors show extremely weak fluorescence due to the unrestricted torsional rotation. In the presence of target analyte, bright fluorescence can be observed because of restricted torsional rotation. In the presence of albumin, the probe exhibits remarkable 400-fold fluorescence enhancement with high selectivity and sensitivity. The probe was successfully applied in the quantitative detection of urinary albumin.

並列關鍵字

fluorescence probe ； cell image ； molecular rotor

參考文獻

17. Yang, Z. G.; Cao, J. F.; He, Y. X.; Yang, J. H.; Kim, T. Y.; Peng, X. J.; Kim, J. S.

20. Liu, T. K., Hsieh, P. Y.; Zhuang, Y. D.; Hsia, C. Y.; Huang, C. L.; Lai, S. P.; Lin, H. S.; Chen, I. C.; Hsu, H. Y.; Tan, K. T. ACS Chem. Biol. 2014, 9, 2359.

8. Ogawa, M.; Kosaka, N.; Choyke, P. L.; Kobayashi, H. ACS Chem. Biol. 2009, 4, 535.

16. Y.-H. Ahn, J.-S. Lee and Y.-T. Chang, J. Comb. Chem. 2008, 10, 376.

1. Bairoch, A. Nucleic Acids Res. 2000, 28, 304.