結合功能性磁奈米粒子之電化學生物感測器於氣喘生物指標之偵測

Abstract

在本研究中，我們開發了偵測氣喘生物指標--嗜酸性白血球陽離子蛋白 (eosinophil cationic protein，ECP) 之電化學生物感測器。先前之研究中證實，支氣管上皮細胞上大量表現的硫酸乙醯肝素 (heparan sulfate) 與類似結構之肝素 (heparin) 對ECP 有專一及高度的鍵結親合力。於本實驗中用來當作檢測ECP之探針。具磁性金包覆鐵奈米粒子 (Au@Fe3O4 NPs) 目前已被證實具有低毒性及良好的生物相容性，被廣泛應用於各種生物樣品分析實驗中。因此，我們使用化學合成方法，利用半胱胺 (cysteamine) 為鍵結的橋樑，將硫酸乙醯肝素的相似物肝素藉由自組裝的方式修飾至 Au@Fe3O4 NPs 上，並利用修飾上肝素的功能性磁奈米粒子 (Hep-Au@Fe3O4 NPs) 添加至含有 ECP 樣品溶液中，使分散於溶液的ECP 會被功能性磁奈米粒子所抓取。此時，在電極後方施與一外加磁場，抓取到 ECP 的功能性磁奈米粒子 (ECP@Hep-Au@Fe3O4 NPs) 即會藉由磁性吸附的關係聚集至電極表面，並增加 ECP 於電極表面的濃度。當增加樣品溶液的體積以及功能性磁奈米粒子的數量，將會增加其電化學感測器偵測 ECP 的靈敏度。 由於 ECP 不具有電化學活性，因此，在本研究中，我們使用 Ru(NH3)63+/2+ 當作電化學活性物質，用以間接偵測 ECP。當溶液中沒有 ECP時，修飾肝素的功能性磁奈米粒子表面帶有高度的負電性，會藉由靜電吸引力的關係，將大量的 Ru(NH3)63+/2+ 吸附至電極表面，產生明顯的Ru(NH3)63+/2+ 氧化還原電流。反之，當溶液中含有 ECP 時，抓取到 ECP 的功能性磁奈米粒子的表面負電荷會因為 ECP 的遮蔽而下降，再加上由於 ECP 的等電位點為 10.8，在中性溶液下帶正電，會排斥溶液中的 Ru(NH3)63+/2+ 擴散至電極表面，降低 Ru(NH3)63+/2+ 氧化還原電流值。當溶液中的 ECP 濃度越高，其電流下降的幅度也越大，因此我們藉由電流下降的差值間接定量溶液中 ECP 的濃度。 本研究所開發的磁性電化學生物感測器不僅具有預濃縮溶液中 ECP 濃度的優點，亦可藉由磁力分離，將 ECP 從複雜的基質樣品中萃取出來，減少大部分的樣品基質干擾。本研究所設計的生物感測器，未來更可以結合至標靶藥物的開發，做更廣泛的應用。

In this study, the electrochemical biosensor combined with functional magnetic nanoparticles for detection of asthma biomarker was developed. Eosinophil cationic protein (ECP) was used as biomarker for asthma, which was been proved that bound with heparin and heparan sulfate expressed on cell membrane specifically. The synthesized magnetic core-shell Au@Fe3O4 NPs existed good bio-compatibility. Based on the specific binding between ECP and heparin and heparan sulfate, the heparin was immobilized on the Au@Fe3O4 NPs using the cysteamine as the linker. The heparin modified Au@Fe3O4 NPs were added into sample solutions containing ECP. After the ECP distributed in solution were captured by heparin modified Au@Fe3O4 NPs, an external magnetic field was applied behind the electrode. The Au@Fe3O4 NPs gathered to the surface of electrode and the ECP concentration near the electrode surface increased. By using this strategy, the sensitivity of designed biosensor could be further improved with larger sample volume used with corresponding amount of nanoparticles added. The Ru(NH3)63+/2+ was used as the electrochemical redox mediator. In the absence of ECP, the heparin modified Au@Fe3O4 NPs carrying large amount of negative charges, which attracted large amount of Ru(NH3)63+/2+ in the solution to provide the electrochemical redox signal. In the presence of the ECP, the heparin modified Au@Fe3O4 NPs bound with ECP and decreased its negative charge. Due to the positive charges carried by ECP, the Ru(NH3)63+/2+ was repulsed from the electrode surface. The redox signal of Ru(NH3)63+/2+ was decreased and the current difference could be used for quantitation of ECP. This designed magnetic nanoparticle based electrochemical biosensor was not only pre-concentrated the ECP concentration, but also extracted the ECP from the sample matrix like human serum or plasma. Most of electrochemical interference issues could be eliminated. This presented method could be further applied into similar probe-target combinations for more extensively usages.