以酵素驅動型核酸分子奈米機器為放大策略發展快速篩檢胰臟癌相關的微小核醣核酸片段之電化學生化感測器

Abstract

雖然胰臟癌的發生事件比起其它癌症來得低，可是它卻是造成癌症相關死亡事件的第四名殺手。由於罹患胰臟癌的病人不會表現出特異的性狀，且胰臟癌的的偵測與診斷存在一定的困難性，因此胰臟癌確診病人的五年存活率只有6%左右。根據先前的文獻指出，罹患上胰臟癌的病人會大量表現某些特定循環性的微型核醣核酸 (microRNAs)，並可在病人血液中被偵測出。在本研究中，我們應用酵素驅動型核酸分子奈米機器為放大策略，發展用於檢測胰臟癌相關之生物標記分子miR-221的電化學生化感測平臺。此平臺主要含蓋三部分，分別為⑴. 聚合酶輔助目標分子之循環反應 (Polymerase-assisted Target Recycling)、⑵. 外切酶剪切反應 (Exonuclease Digestion) 和⑶. 電化學訊號的輸出 (Electrochemical Signal Output)。我們首先以膠體電泳的方式先進行產物初步之分析，證明當受質Hairpin (H)、目標分子miR-221 (M)、聚合酶 (P)、外切酶 (E) 四大元件缺乏其一時，DNA Nanomachine則無法運作，最終產物P2就無法成功產出。接著我們進行一系列條件之最佳化，其中包括緩衝溶液的選擇、受質使用的濃度、聚合酶和剪切酶的濃度比，以獲得最多的最終產物P2。在電化學偵測的部分，我們先確認最終產物P2可以成為誘導股，並與修飾在工作電極表面亞甲藍標定的探針進行雜合，進而造成電化學訊號之改變。接著我們進行阻隔劑 (Blocking agent) 和鍍金時間之最佳化，以得到最佳之實驗操作條件。藉由標準曲線 (Calibration curve) 的結果顯示，我們所設計的電化學生化感測器的偵測極限為9.6 pM。我們將20 fmol的miR-221添加入血清中，所測得之回收率 (Recovery rate) 接近百分之百。該感測平台被證實具有可偵測血清中之microRNA之可行性和潛力。此外，我們也使用不同分析物 (目標分子和其同源家族成員) 進行DNA Nanomachine之選擇性測試，結果顯示所研發的電化學感測器只會針對目標分子miR-221展現出訊號之改變，從而證實我們所研發的電化學感測器具有良好之選擇性。

Pancreatic cancer with a single-digit survival rate, at ~7%, has the highest mortality rate among all cancers. Similar to lung cancer, pancreatic cancer patients seldom exhibit disease-specific symptoms until late disease stage; therefore an establishment of early detection method for pancreatic cancer is important to classify pancreatic cancer at earlier stages. MicroRNAs (miRNAs) are a class of highly conserved non-coding RNA consisted of approximately 22 nucleotides that regulate gene expression by either degradation of mRNAs or translational repression at the post-transcriptional level. MicroRNAs are voted as potential biomarkers for therapeutics and point-of-care diagnostics, as their aberrant expressions are always associated with diseases and even various cancers. In this study, a novel isothermal electrochemical biosensor was designed for sensitive and selective detection of circulating pancreatic cancer-related microRNA. The amplification strategy was based on dual enzyme-empowered target recycling performed by polymerase and exonuclease. Initially, the presence of target miRNA selectively unfolded a hairpin probe (H), followed by the polymerase-triggered elongation of the H along with the strand displacement of target miR. While the target miR went on a new cycle of H unfolding, a blunt end of the He was formed and digested by the exonuclease, resulting in the release of intermediate DNA product (P1). The released P1 was able to hybridize with a new H, initiating a new round of exonuclease-assisted digestion of H, yielding a final DNA product (P2). Finally, a “signal-off” electrochemical biosensor was established when the signal inducer P2 hybridized with a methylene blue-labelled complementary hairpin probes (P2C) that immobilized on a gold nanostructured screen printed electrode (AuNC@SPCE) through gold-thiol bond. Under optimal condition, this biosensor offered an excellent sensitive platform towards the detection of target miR, with detection limit of 25 pM together with a dynamic range of six orders of magnitude. In addition, an excellent selectivity was achieved in discriminating its homologous inter-family members. We have herein successfully demonstrated a biosensing platform for the detection of pancreatic cancer-related miR and the biosensor holds a potential in the early diagnosis of pancreatic cancer to improve the survival rate in the future.