奈米金增強電化學訊號於腸病毒71型快速高靈敏檢測系統之開發

Abstract

腸病毒71型(Enterovirus 71) 為一新興傳染病，在感染之後出現發燒和發疹後的二至三天，能造成神經方面的後遺症及死亡。診斷腸病毒71型是非常困難的。因此，在臨床上需要一個快速、高靈敏度、高專一性和操作方便的檢測腸病毒71型的方法。 電化學頻譜儀，可以偵測工作電極表面的分子反應，偵測簡易、快速而且敏感。電化學頻譜儀已廣為使用在偵測蛋白質、核糖核酸及去氧核糖核酸。而抗原和抗體的反應可以提高偵測的專一性。此外，奈米金可以藉由增加表面積、降低阻抗和避免抗原和抗體作用時產生的立體障壁來增強電化學頻譜儀的訊號。三種方法的結合使腸病毒71型的檢測可以快速、高靈敏度、高專一性和操作方便。 之前對電化學頻譜儀的研究中，鮮少有討論到工作電極之製程與偵測能力之關係。本研究以不同之介質層和不同大小之奈米金顆粒對腸病毒71型病毒的偵測能力的影響來進行研究。三種不同的自組裝單分子層：半胱胺-戊二醛-半胱胺(cysteamine-glutaraldehyde-cysteamine，CGC)；三巰基丙胺( 3-mercaptopropionic acid (3-MPA))；3-巰丙基三甲氧基硅烷((3-Mercaptopropyl) trimethoxysilane (3-MPS))，被選擇用來評估自組裝單分子層對阻抗的影響。為使更微量的腸病毒71型顆粒能被偵測，因此選用低阻抗的介質層。我們選擇CGC為中間之介質層。因為其結構具有雙鍵，故電阻最低，只有0.7 Kohm。此外與3-MPS比較，CGC無須強鹼脫水，比較環保。另外我們也比較了2、18與40奈米(nm)之奈米金顆粒對電極偵測能力之影響。發現18nm之奈米金有最好之偵測極限及偵測範圍。 藉由此製程之奈米金顆粒修飾之電化學頻譜儀探針來偵測腸病毒71型，我們的結果顯示，這個檢測探針不但可以區分腸病毒71型與克沙奇A16 (Coxsackievirus A16)、單純皰疹病毒(Herpes Simplex Virus)和溶菌酶(Lysozyme)，其靈敏度可達1 copy number / 50 microl，還具有定量的功能，其偵測範圍為1- 6050 copy numbers / 50 microl。從樣品浸泡到電化學頻譜儀偵測的時間只需約11分鐘。因此此一檢測平台具有直接在臨床醫療使用之潛力。

Enterovirus 71 (EV71) infection is an emerging infectious disease causing neurological complications and/or death within two to three days after the development of fever and rash. Clinically, diagnosis of EV71 infection is very difficult. Therefore, a rapid, highly sensitive, highly specific, and user-friendly test is needed. Electrochemical impedance spectroscopy (EIS), which measures the molecular interaction on the surface layer of the working electrode, is rapid, sensitive and easy operated. EIS has been widely used in detection of protein, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Additionally, nanogold particles (AuNPs) can enhance the signal of EIS by increasing the surface area and reducing impedance and steric hindrance between antigen (Ag)–antibody (Ab) interaction, thus increasing the sensitivity of EIS. Furthermore, it is well known that antigen and antibody binding is highly specific. Application and combination of these methods for EV71 detection satisfied the requirement of rapidity, high sensitivity, high specificity, and friendly operation. Conventional EIS studies do not focus on the relationship between the interfacial layer of the working probe and the detection capability. For improving EV71 detecting performance, this study systematically evaluated the interfacial layers with various thiol-linkaged polymers and the sizing effect of nanogold particles (AuNPs). Three different self-assembled monolayer: cysteamine-glutaraldehyde-cysteamine (CGC), 3-mercaptopropionic acid (3-MPA), and 3-Mercaptopropyl) trimethoxysilane (3-MPS), were chosen for testing the impedance changes of EIS. For EIS detection lower charge transfer resistance (Rct) of interfacial layer is highly desired. Owing to the double bond resonance in the structure of CGC SAM, it has the lowest Rct (0.7 Kohm). In spite of lowest Rct, the CGC SAM formation procedure is relative environmental friendly than 3-MPS SAM formation. Furthermore, 2 nm, 18 nm and 40 nm nanogold conjugated working electrode has the different detection limit and range. Eighteen nm nanogold conjugated working electrode has the best detection limit and range (detection limit 1 copy number/50 microl, detection range 1-6060 copy number/50 microl). We developed an EV71-specific nanogold-modified working electrode for electrochemical impedance spectroscopy in the detection of EV71. Our results show that the probe can distinguish EV71 from Coxsackievirus A16 (CA16), Herpes simplex virus (HSV), and lysozyme. The detection limit of EV71 is as low as 1 copy number/50 microl reaction volume and its linear range is from 1 to 6050 copy numbers /50 microl reaction volume. It takes less than 11 minutes in sample preparation and EIS detection. This detection platform may have the potential for use in point-of-care diagnostics.