标题: | 建构与运用谷胱胺肽生物感测试片 Fabrication and Application of Glutathione Biosensing Strips |
作者: | 江品萱 林志生 分子医学与生物工程研究所 |
关键字: | 生物感测器;谷胱胺肽;奈米金球;网版印刷碳电极;酪胺酸酶;Biosensor;Glutathione;Gold nanoparticles;Screen-printed carbon electrode;Tyrosinase |
公开日期: | 2011 |
摘要: | 谷胱胺肽(glutathione; GSH)是生物体内一重要的抗氧化物质。它不只可以直接清除自由基,还可以增强其他重要抗氧化剂的能力,例如:维生素C和维生素E。现今有许多研究显示,GSH浓度的改变和癌症、阿兹海默症、帕金森氏症、糖尿病、黄斑病变、后天免疫不全症候群(AIDS)等疾病有相关。然而,传统上侦测GSH的方法有许多限制,例如检测时间较长、有些样品需要做前处理及缺乏足够的灵敏度等。本研究的目的为建构一可携带且一次性使用的酪胺酸酶(tyrosinase)网版印刷碳电极(screening-printed carbon electrode; SPCE)生物感测试片,并将此试片应用在细胞和动物检体中GSH浓度的检测。 此GSH检测系统以酪胺酸酶做为生物辨识元件,并藉着安培式讯号转换器将SPCE感测试片的讯号转换。我们先将试片的工作电极与参考电极上修饰氨水(ammonium hydroxide; NH4OH),再修饰奈米金球(colloidal gold nanoparticles; AuNPs)与介电子二茂铁(ferrocendicarboxylic acid; FeDC)于工作电极和参考电极表面。AuNPs的功用为提供电子在氧化还原酵素与电极间的传递,及提供生物分子(酵素)在固定化时一个稳定的电极表面,以维持生物分子的活性。介电子FeDC可帮助生物分子的氧化还原反应并做为一个电子梭子,加速酵素及电极间的电子传递以提升讯号。最后将酪胺酸酶固定在SPCE感测试片上,再利用循环伏安法探讨AuNPs及介电子在此生物感测系统的特性,经结果可以得知在AuNPs以及FeDC的加成作用下,其循还伏安法的还原波峰电流放大5.2倍。 在此系统中使用邻苯二酚(catechol)为酪胺酸酶的受质。当邻苯二酚与试片上的酪胺酸酶氧化生成醌(o-quinone)后,电极提供一低电压使醌还原成邻苯二酚。其中影响此反应的参数包括pH值、缓冲液浓度及操作电压。此系统之最佳化条件为pH值为6.5、缓冲液浓度为50 mM与-200 mV的电压。当在-200 mV的电压下进行反应时,可在40秒内获得最大的反应电流。此外,在最佳化条件下,反应电流与邻苯二酚浓度呈正相关且浓度线性范围为0到1000 uM。 此系统再现性测试方面,在血清中加入不同浓度的GSH,用此生物感测系统检测可以得到极佳的再现性。此外,长时间稳定性测试方面,将修饰好的SPCE长时间保存在4°C下,可以维持84%至少六周的活性,由结果显示,此酪胺酸酶修饰之生物感测试片可以长时间稳定的保存在4°C下且不会失去活性。 将酪胺酸酶修饰之生物感测试片应用在细胞与动物的GSH浓度检测上,可以成功地检测动物血液、血清及器官中和细胞中的GSH浓度。利用GSH抑制醌在酵素及电极间的循环,GSH和醌生成GSH-醌 (glutathione-quinone) 复合物,导致还原电流降低。使用酪胺酸酶生物感测试片测得的GSH线性浓度范围为32.5到500 uM。 此酪胺酸酶修饰之电化学生物感测系统可以成功的做为一个即时检测的安培量测系统。此研究证实,利用AuNPs和FeDC修饰酪胺酸酶生物感测试片具有相当的潜力于临床GSH的样品检测。 Glutathione (GSH) is a major antioxidant in organisms. It not only detoxifies reactive oxygen species directly, but also enhances the functional ability of other crucial antioxidants, such as Vitamins C and E. Alteration in GSH concentration has been implicated in a number of pathological conditions, including cancer, Alzheimer’s, Parkinson’s diseases, diabetes, macular degeneration, acquired immune deficiency syndrome (AIDS) and HIV disease. However, GSH detection has always proven to be difficult. Most of the methods are time consuming, needing derivatization and lack sufficient sensitivity. In this study, the disposable and tyrosinase-modified electrochemical test strips were fabricated for the detections of GSH level in vitro and in vivo. The system was based on tyrosinase as biorecognition element and screen-printed carbon electrode (SPCE) as an amperometric transducer. The bilateral modification of the electrodes was utilized because the method had higher current compared to the modified unilateral electrode. At first, the working and reference electrodes of the SPCE strips were immobilized by ammonium hydroxide (NH4OH). The strips were modified with colloidal gold nanoparticles (AuNPs) and mediator (ferrocendicarboxylic acid; FeDC). The AuNPs not only permit electron transfer between redox chemicals directly and bulk electrode materials, but also provide a stable surface for the immobilized biomolecules retaining their biological activities. The immobilized mediator promotes the redox reactions of biomolecules and shuttles the electrons more efficiently between tyrosinase and electrodes, resulting in enhancing the sensing performance. Finally, tyrosinase was immobilized on the surface of working electrode of SPCE strips. The electrochemical performance of the modified SPCE strips was characterized by cyclic voltammetry. In the presence of AuNPs and FeDC, the cathodic peak current of the cyclic voltammogram was significantly enhanced by 5.2 folds. In the tyrosinase-SPCE strips, catechol was used as the substrate of tyrosinase. The oxidation reaction from catechol to o-quinone catalyzed by the immobilizing tyrosinase. This enzymatically generated o-quinone could be reduced electrochemically back to catechol by providing with a low potential and the reduction current was measured to determine the quantity of substrate. Factors affecting the response current, including pH, phosphate buffer concentration and working potential were investigated. The optimum pH was 6.5 and the optimum concentration of phosphate buffer was 50 mM. The tests were performed in the presence of the catechol substrate at -200 mV. The maximum response from the oxidation reaction of o-quinone was obtained within 40 sec. Under the optimized condition, the response was found to be linear and concentration-dependent in the range to 0 to 1,000 uM of catechol. The detection mechanism of GSH in the above-samples was accomplished by suppressing the recycling process which occurs between tyrosinase and electrode of biostrip. The GSH reacted with o-quinone and glutathione-quinone complex was produced. The production of glutathione-quinone complex reduced the quantity of o-quinone and made the reduction current decreasing. The linear response of the tyrosinase-SPCE strip was ranging from 32.5 to 500 uM of GSH. For testing the system stability and recovery for GSH determination in biological samples, the plasma samples were then spiked with different concentrations of GSH and the recovery values were calculated. For all the concentrations tested (200, 400, 600, 800, and 1000 uM of GSH), good recovery values were obtained. The results demonstrate that the thiol–disulfide exchange reaction is not influenced by the presence of a complex matrix. Both the time and completion of the reaction are similar of those obtained in standard solutions. GSH was tested in the concentration range which could be found in normal and pathologic conditions, with no substantial differences in the recovery values. In long-term storage stability of the modified-SPCE strips, the prepared tyrosinase SPCE strips were stored at 4°C; it retained 84% of its initial response at 6th week. It means that the developed tyrosinase-SPCE strips were stable for several weeks if they are stored at 4°C. The tyrosinase-SPCE strips were applied in biomedical experiments. In the experiments, the mice were challenged with doxorubicin and administrated with N-acetylcysteine or astaxanthin. After 14 days of treatment, the mice were scarified and the blood and tissues, including heart, lung and liver, were sampled for the detection of GSH levels. The results show that doxorubicin treatment could reduce the blood and visceral GSH level and N-acetylcysteine and astaxanthin could compensate this physiological GSH depletion. In conclusion, a disposable amperometric biosensor, the tyrosinase and AuNPs modified SPCE strips were fabricated. The strips were successfully utilized to real-time detect the GSH level in biological samples indicating that the strips are potential of forward applications in GSH detection for clinical diagnosis. |
URI: | http://140.113.39.130/cdrfb3/record/nctu/#GT079929503 http://hdl.handle.net/11536/49974 |
显示于类别: | Thesis |