食品中黃麴毒素B1檢測用適體之篩選

黃麴毒素（aflatoxin B1, AFB1）為一種由真菌所產生的毒素，常常出現在保存不當的食品中，具有強烈的肝毒性與致癌性；有鑑於大多國家皆使用成本高且耗時的高效液相層析儀（high performance liquid chromatograph, HPLC）作為檢測方式，故有必要開發快速的AFB1感測器；近年來，利用核酸分子進行感測技術逐漸成熟，其中以具有高親和力與高選擇性之適體（aptamer）最為大宗，為了篩選出與開發感測器之實驗條件相同之適體，本研究以磁性奈米粒子（magnetic nanoparticle, MNP）與磁性輔助篩選適體平台（magnetically-assisted rapid aptamer selection, MARAS）來篩選AFB1適體，並利用即時定量聚合酶連鎖反應法（real-time quantitative polymerase chain reaction, Q-PCR）衡量其解離常數（dissociation constant, Kd），選擇結果中表現較佳的適體作為感測器之辨識分子，供往後製備電化學阻抗式適體感測器之用；此感測器基於柱狀金電極和電化學阻抗頻譜法（electrochemical impedance spectroscopy, EIS），將適體藉由硫醇基固定於金電極表面，便可透過量測表面阻抗變化來感測AFB1之濃度，最終評估與比較MARAS篩選適體與文獻中適體之優劣以及感測器開發潛力；在MARAS實驗後，共篩選出六條適體，經過即時定量聚合酶連鎖反應法評估後，選擇親和力表現最強的一條適體，其解離常數量測結果為58.93nM，由同樣方法量測文獻適體之解離常數為188.7nM，並由此適體與文獻適體開發成阻抗式感測器，兩者對於AFB1感測之檢量線均呈線性關係，且經過推導計算，兩者在阻抗式感測器中對AFB1結合之解離常數分別為43.67nM（MARAS適體）與55.56nM（文獻適體）；由此推論MARAS不但大幅縮短了原本篩選適體時繁瑣的實驗步驟，且篩選之適體與文獻中之適體對AFB1具有相同程度之親和力，故適合作為開發適體感測器之篩選技術。

關鍵字

磁性奈米粒子；磁性輔助篩選適體平台；即時定量聚合酶連鎖反應法；解離常數；電化學阻抗頻譜法；適體感測器

並列摘要

Aflatoxin B1 (AFB1) is a potential food pollutant produced by fungi during inappropriate storage environment. Because of strong hepatotoxicity and carcinogenicity, many countrys have established restrict regulation for residue of AFB1 in foodstuffs and use high performance liquid chromatography (HPLC) to detect AFB1. However, due to some disadvantage of HPLC, including high cost and time-consuming procedures, it is necessary to develop a simple and highly sensitive biosensor for the detection of AFB1. In this work, magnetic nanoparticles (MNP) and magnetically-assisted rapid aptamer selection (MARAS) were used to effectively screen DNA aptamers that recognize AFB1. Real-time quantitative polymerase chain reaction (Q-PCR) was measurement method to calculate dissociation constant (Kd), which evaluate the binding affinity between aptamer and analyte. In order to discuss feasibility of biosensor based on aptamer from MARAS, an aptasensing device has been designed by gold electrode and electrochemical impedance spectroscopy (EIS) for determination of AFB1 concentration. After MARAS, we selected one aptamer with highest affinity by Q-PCR, its dissociation constant was determined to be 58.93nM. Compared to patented aptamer for AFB1, the dissociation constant was 188.7nM. Both designed aptasensor of MARAS aptamer and patent aptamer exhibited linear range from 1 to 100ppb with a coefficient correlation of 0.9613 and 0.9817. After analysis, the dissociation constant of MARAS aptamer was 43.67nM, and patent aptamer was 55.56nM, calculated by EIS. Based on similar dissociation constant, the MARAS experiment efficiently generates aptamers with high affinity to AFB1 but consumes fewer resources and time. Furthermore, due to elasticity and customization of experimental variables, MARAS is completely suitable for research and development of aptasensor.

並列關鍵字

Magnetic nanoparticle ； Magnetically-assisted rapid aptamer selection ； Real-time quantitative polymerase chain reaction ； Dissociation constant ； Electrochemical impedance spectroscopy ； Aptasensor