發展大腸桿菌O157、維生素H及生物科技作物 之電化學感測平台

Development of Electrochemical Sensing Platforms for the Detection of Ecoli O157, Vitamin H and Biotech Crops

廖韋晴(Wei-Ching Liao)

指導教授 ： 何佳安 ； 陳益佳

國立清華大學/理學院/化學系/博士(2013年)

摘要

在臨床、環境和生物工業領域，為了達到即時監督及檢測的目的，發展快速、靈敏、可攜式且低成本的生物感測器是重要且迫切需要的。本論文分別針對 (一)食物中常見的致病菌大腸桿菌O157、(二) 生理中維持健康的維生素H及 (三) 生物科技產物基因改造作物，發展了三種電化學生物感測系統。
腸溶血性大腸桿菌O157，會生成類志賀毒素 (Shiga-like toxins, verocytotoxin) 造成急性或慢性腎功能衰竭而導致死亡。為加快臨床檢驗的速度，在第一部分，我們發展一新型的電化學基因感測器，以電沉積法製作奈米金網版印刷電極，接著以硫–金鍵結自組裝的方式修飾核酸探針於電極上，對大腸桿菌O157特有的rfbE基因進行感測。運用競爭型分析方法的原理，結合包覆電化學訊號分子三氯化六銨合釕的微脂體 (hexaammineruthenium(III) chloride-encapsulated liposomes) 進行訊號放大，最後以電化學方波伏安法偵測由微脂體釋放的電化學分子訊號。實驗結果可得一線性範圍1–106 fmol的檢量線，對大腸桿菌rfbE基因的偵測極限為0.75 amole，相當於體積5 μL濃度0.15 pM的分析樣品，與許多已報導的文獻相比，本方法具有更佳的偵測極限。
一般的抗體修飾方法可會造成抗體結構改變或失去活性。為了改善此缺點，並增進免疫分析系統對目標物的感測能力，在第二部分中，我們利用3-噻吩硼酸(thiophene-3-boronic acid) 親和力製作具有位向性的抗體修飾之奈米金網版印刷電極。將3-噻吩硼酸以自組裝方式修飾於奈米金網印電極，藉由硼酸與二元醇之間的親和力，將抗體 (含有二元醇的醣蛋白) 固定於電極上，並利用硼酸將抗體接合在電極上會使抗體具有方向性，增加免疫電極的感測效能。實驗中，以維生素H為分析物，與表面鍵結維生素H且包裏電化學分子六氰亞鐵酸鉀的微脂體 (potassium hexacyanoferrate(II)–encapsulated liposomes)，進行競爭型免疫分析。經電化學方法及表面電漿共振實驗分析，證實硼酸親和力修飾之免疫感測器，能達到抗體位向性修飾、保持抗體活性及增進感測效能。
第三部分為建構基因改造作物的數位分子邏輯閘感測平台。因為生物技術的發展，市面上已有許多基因改造作物產品，為保障消費者購買時具有選擇的權力，各國分別制定相關的基改食品標示及管理法規。為了能夠快速精確地檢驗食品中的基改成份，我們運用核酸分子的序列專一性及酵素梯瀑 (enzyme cascade) 的訊號傳遞方式，結合邏輯運算法則，發展出不同於以往的基因改造作物檢測平台。若以歐盟訂定的0.9 %基改食品標示準則，此系統目前能分析至少25 nM的樣品濃度，能有效地對基因改造作物進行定性及定量分析。
透過多樣的感測原理配合新穎的生物技術，期望在疾病診斷、環境監控或居家重點照護各方面，發展出更便捷、精準及人性化的生物感測器，以改善人類的生活品質。

關鍵字

電化學 ； 去氧核醣核酸 ； 生物感測器 ； 微脂體 ； 邏輯閘 ； 抗體位向性修飾

並列摘要

Development of rapid, sensitive, portable, and low cost biosensors is important and in urgent need in clinic, environmental, and bio-industrial fields for real-time monitoring and diagnosis. In this dissertation, three electrochemical biosensing platforms are designed and demonstrated for the detection of pathogenic Escherichia coli O157, dietary supplement of vitamin H, and biotech product–genetically modified crops.
Enterohemorrhagic Escherichia coli O157, a verocytotoxin-producing pathogen, can be deadly because it can induce acute or chronic renal failure. To speed up the clinical diagnosis of related syndromes caused by E. coli O157, we have developed a novel electrochemical genosensor, featuring nanogold-electrodeposited screen-printed electrodes modified with a self-assembled monolayer of thiol-capped ssDNA capture probe, for the detection of the rfbE gene, which is specific to E. coli O157. Based on the mechanism of competition assay and signal amplification through hexaammineruthenium(III) chloride-encapsulated liposomes, the current signal of the released liposomal Ru(NH3)63+ was measured using square wave voltammetry, yielding a sigmoidally shaped dose−response curve whose linear portion was over the range from 1 to 106 fmol. We could detect as little as 0.75 amol of the target rfbE DNA (equivalent to the amount present in 5 μL of a 0.15 pM solution), which is much more sensitive than those reported previously.
In the second part of this dissertation, a new immobilization strategy for site orientation of the antibody using thiophene-3-boronic acid (T3BA) has been demonstrated for improving the sensing performance. The immunosensor comprised a nanogold-electrodeposited screen-printed electrode modified with a self-assembled monolayer of T3BA. Anti-biotin (vitamin H) antibody (a glycoprotein) was covalently bound to T3BA through boronic acid–saccharide interactions. The assay functioned based on competition between the analyte biotin and biotin-tagged potassium hexacyanoferrate(II)–encapsulated liposomes. The current signal produced by the released liposomal Fe(CN)64– was measured using square wave voltammetry. In this proof-of-concept study, as confirmed through electrochemical and surface plasmon resonance (SPR) analyses, this T3BA approach (i) enables site orientation of the antibody molecules, (ii) maintains the activity of the captured antibodies, and (iii) significantly improves the assay performance.
Finally, in the third part, we constructed a biomolecular logic gate system to digitally identify genetically modified organisms (GMOs). Because of the advances in biotechnology, GMOs have been well developed and commercialized in many countries for the past decade. In order to provide consumers efficient information of genetically modified (GM) food, various countries (e.g. EU) have implemented labeling regulations and safety evaluation systems for management of GM foods. To fulfill the GMO concerning legislation, reliable and sensitive methods to detect GMOs in foods are necessary. We have demonstrated a biomolecular logic gate (circuit) system with computing functionality mimicking the generation process of an event-specific gene and its feasibility in the analysis of GMOs to provide an alternative to the complex procedures commonly involved in the screening of GMOs.
Overall, the combination of various assay formats (competitive assay or sandwich assay), detection platforms (electrochemical or colormetric), nanofabrication technique (carbon electrode constructed with nanogold), powerful signal amplifiers (liposome and enzyme) and biologic gate processing (AND gate), we have successfully developed three simple, accurate, and humanized biosensors that exhibit the feasibility of being extended to the point-of-care diagnostics, environmental monitoring, and food safety surveillance.

並列關鍵字

electrochemistry ； DNA ； biosensor ； liposome ； logic gate ； site-oriented immobilization of antibody

參考文獻

141. Livaniou, E.; Costopoulou, D.; Vassiliadou, I.; Leondiadis, L.; Nyalala, J. O.; Ithakissios, D. S.; Evangelatos, G. P., Analytical techniques for determining biotin. J. Chromatogr. A 2000, 881 (1-2), 331-343.

51. Maurer, J. J.; Schmidt, D.; Petrosko, P.; Sanchez, S.; Bolton, L.; Lee, M. D., Development of primers to O-antigen biosynthesis genes for specific detection of Escherichia coli O157 by PCR. Appl. Environ. Microbiol. 1999, 65 (7), 2954-2960.

1. Wang, J., Towards genoelectronics: Electrochemical biosensing of DNA hybridization. Chem. Eur. J. 1999, 5 (6), 1681-1685.

2. Patolsky, F.; Lichtenstein, A.; Willner, I., Electronic transduction of DNA sensing processes on surfaces: amplification of DNA detection and analysis of single-base mismatches by tagged liposomes. J. Am. Chem. Soc. 2001, 123 (22), 5194-5205.

3. Patolsky, F.; Lichtenstein, A.; Willner, I., Detection of single-base DNA mutations by enzyme-amplified electronic transduction. Nat. Biotechnol. 2001, 19 (3), 253-257.