開發銀奈米團簇之生醫感測器偵測大腸直腸癌之肝轉移

Abstract

研究指出超過百分之五十的大腸直腸癌患者會有癌細胞轉移至肝臟的情形發生，轉移導致這些病患的存活機率小於未發生癌症轉移的病患，然而有肝轉移的病患若能提早接受手術切除肝腫瘤以及合併使用化學藥物進行輔助治療則可以顯著提升病患的存活率。因此，早期偵測並正確找出具有高轉移風險的病人將有效提升病人的痊癒機率，減少潛在的危險。 先前研究也證實小分子核醣核酸miR-885-5p可作為大腸直腸癌之肝轉移的生物標記。於此，我們開發了一個針對異常表現之miR-885-5p的偵測平臺，本平台中包含兩個系統，分別為由miR-885-5p誘導啟動之去氧核酶 (DNAzyme)與裝載於去氧核醣核酸奈米環 (DNA nanoring)的銀奈米團簇 (DNA-templated sliver nanoclusters, AgNCs)，我們稱之為DNA nanoring@AgNCs。由miR-885-5p所活化之去氧核酶 (DNAzyme) 可以剪切溶液中的核醣核酸-去氧核醣核酸受質並產生兩條單股去氧核醣核酸，其中的一條我們稱之為convertor。Convertor可進一步以鏈置換反應 (Strand displacement)促使銀奈米團簇產生紅色螢光，以此做為訊號輸出。 在本研究中我們針對AgNCs的緩衝溶液條件、反應時間及溫度進行優化，使AgNCs螢光與鏈置換反應皆有最好之表現，同時成功合成DNA nanoring@AgNCs之奈米結構，並以穿透式電子顯微鏡加以驗證。而在DNAzyme的部分則以電泳的方式確定DNAzyme只在miRNA存在時得以活化並剪切受質產生convertor序列，最後確認兩系統可以合併使用，完成本偵測平臺。 本研究未來將著重於提升偵測平台之應用性以針對病患之血液檢體進行分析，同時探討利用DNA nanoring裝載AgNCs於生醫分析平臺上的更多應用性。

More than fifty percent of patients with colorectal cancer (CRC) will undergo liver metastasis, which ultimately results in relative low survival rate compared with those who with primary cancer. Early detection, searching for effective prognostic indicators of treatment response and accurate identification of patients at high risk for recurrence are believed to be useful strategies to improve cancer outcomes. The circulating miRNA-885-5p was found to be a promising prognostic biomarker for detect hepatic metastasis from colorectal cancer. We herein developed a miRNA-sensing platform, integrating a miRNA-activated DNAzyme amplification system and DNA-templated silver nanoclusters (AgNCs) conjugated on a DNA nano-ring (DNA nanoring@AgNCs) for sensitive detection of miRNA-885-5p. The DNAzyme first hybridized with the locker strand and remained inactivated until the presence of our target miRNA. The target miRNA removed the locker strand from the DNAzyme via strand displacement to re-activate DNAzyme. The DNAzyme possess the catalytic ability to cleave specific DNA-RNA substrates introduced in the reaction mixture, and subsequently produced two short single strands DNA, which served as signal convertor for our sensing platform. The convertor DNA triggered the transformation of green-emissive AgNCs to red-emissive AgNCs, which can be fabricated in situ by the hybridization of a specially designed DNA template with a DNA nanoring. We have obtained the optimized reaction conditions for DNA-templated AgNCs, including working buffer condition, reaction time and temperature, to reach high luminescence with AgNCs, and enhanced strand displacement performance. We also hybridized the DNA-templated AgNCs with partially complementary nanoring to form the “DNA nanoring@AgNCs” and it was subjected to observation under High Resolution Transmission Electron Microscope (HRTEM). In addition, we confirmed that the DNAzyme cleavage of the substrate only occurred when target miRNA presented in the sample solution. Last but not least, we successfully combined two systems (AgNCs reporting probe system and miR-885 activated DNAzyme amplification system) into a functioning miRNA-sensing platform for monitoring hepatic metastasis from colorectal cancer prognosis. Further study will investigate not only the applicability of our sensing platform in analyzing real blood samples collected from cancer patients, but also the feasibility of DNA nanoring as a potential signal amplifier in sensing platform.