開發磁性奈米粒子輔助之電化學生化感測器

Abstract

四氧化三鐵磁性奈米粒子 (Fe3O4 magnetic nanoparticles，Fe3O4 MNPs) 具有超順磁性，能夠經由外加磁場的方式簡單操作，且在表面包覆上金奈米粒子後所形成的磁奈米複合材料可廣泛應用在生醫相關的研究上。利用此複合材料含有高生物相容性之特性使其易修飾各種生物探針於表面，可應用於多種包含電化學偵測等不同領域。利用磁性奈米粒子的特性，於本篇論文中將延伸出兩個相關應用。 第一個磁性奈米材料之應用主要是利用一段對 Hg2+ 具有高度專一性結合之 DNA 探針修飾於磁性奈米粒子上以製作一個對於偵測 Hg2+ 具有高度靈敏度的電化學生物感測器。由於胸腺嘧啶和 Hg2+ 之間具有特異性相互作用，故在生物感測器上利用七對胸腺嘧啶與 Hg2+ 做結合而設計一個可以有效偵測 Hg2+ 的感測器。第二個應用主要是用來偵測攝護腺癌，攝護腺癌是目前在男性族群中一種常見的惡性腫瘤，在近年來在男性人口中並列入十大主要癌症死亡原因之一。攝護腺特異性抗原 （prostate specific antigen，PSA） 是一種主要由攝護腺上皮所產生的醣蛋白，為目前最常見的攝護腺癌的血清標誌物，當一般的男性其體內血清含有 PSA 的濃度如果高達 4.0 ng/mL 以上，即認為是陽性結果，有必要再做進一步診斷與治療。在此實驗研究中，主要是利用 PSA 適體對 PSA 的專一性結合電化學製作成生物感測器去偵測人體血清中的 PSA 含量。利用已修飾 PSA 適體於磁性奈米粒子結合滾環式擴增法 (rolling circle amplification，RCA) 及生物條碼 DNA ，可以達到高度靈敏度的生物感測器。在沒有 PSA 的情況下，與 PSA 適體互補的 DNA 片段可以作為 RCA 反應的引子使用。 而利用 RCA 反應可以產生富含大量鳥嘌呤之生物條碼的序列當作放大訊號的機制，而由於大量鳥嘌呤之生物條碼的序列可以特異性的結合亞甲藍電化學訊號物質，故在電化學測量提供了重要的氧化還原訊號，當 PSA 存在時會減少鳥嘌呤之生物條碼序列，進而使得氧化還原訊號下降，藉由此下降量可以有效得知 PSA 在人體中的含量。本實驗所利用的生物感測器具有高選擇性及高靈敏度，在未來可以做為更廣泛的應用。

A sensitive electrochemical biosensor based on Fe3O4 magnetic nanoparticles (Fe3O4 MNPs) existing superparamagnetic characteristic can be easily manipulated by external magnetic field. After coating with gold nanoparticles, the functional magnetic composites can be widely used in biochemical or clinical related researches. The fabricated magnetic nanoparticle was easily to modify with DNA probe to be excellent biocompatibility. Using the function of magnetic nanoparticles, we have two applications in this thesis. The first application was a sensitive biosensor for Hg2+, which was developed based on highly specific designed DNA modified magnetic nanoparticles. Due to the specific interaction between thymine and Hg2+, an artificial DNA sequence was designed with 7 thymine pairs for Hg2+ binding. This method could apply on biosensor for detection of Hg2+. Another application was prostate specific antigen electrochemical biosensor development. Prostate cancer is a common malignancy and listed in top 10 leading causes of cancer death in the male population. Prostate specific antigen (PSA) with molecular weight 33 kDa glycoprotein produced primarily by prostate epithelium and used as biomarker for diagnosing prostate cancer. A serum PSA measurement above a value of 4.0 ng/mL was generally regarded as positive and might indicate the need for a biopsy. In this study, an aptamer based electrochemical biosensor was presented. By using aptamer immobilized magnetic nanoparticles combined with RCA and barcode DNA, an extreme high sensitivity was archived. In the absence of PSA, complementary DNA bind with aptamer and used as primer of RCA reaction. The product of RCA contained G-rich bases and specifically bind with methylene blue, which provide significant redox signal in electrochemical measurement. In the presence of PSA, aptamer molecules fold and bind with PSA, lead to the reducing of RCA primer and results in the decrease of methylene blue redox signal. The current difference of the methylene blue can reflect the concentration of the analyte. The proposed method provides an alternative approach for ultrasensitive detection of aptasensor which has good sensitivity and selectivity.