生物條碼應用於人類免疫球蛋白E電化學生化感測器

Abstract

免疫球蛋白 E (immunoglobulin E，IgE) 在許多自體免疫疾病及過敏症都扮演相當重要的角色。一般來說，我們可以藉由測量血漿或血清中免疫球蛋白 E 總量的濃度去觀測患者的狀況。由於免疫球蛋白 E 的產生是過敏原對個體反覆刺激的結果，因此當體內有高濃度的免疫球蛋白 E 時，常和過敏反應有關。也因為這樣，測量免疫球蛋白 E 總量在臨床上除了可以提供體內過敏反應的訊息外，也可以幫助醫師了解病患目前特異性過敏的情況，以利醫師之後選擇正確的投藥與治療。但是免疫球蛋白 E 在人體血液中的含量極低，在一般的情況下，免疫球蛋白 E 的含量在 100 IU/mL 以下 (約 1.26 nM)。綜合以上的原因，發展簡單、快速且高靈敏偵測免疫球蛋白E的方法是有其必要性的。 在本實驗中使用的電極基材為網版印刷碳電極 (screen printed electrode，SPE) 以亞甲基藍作為電化學訊號的來源。利用定電位安培法的方式將奈米金粒子電沉積在網版碳電極的表面，並將可以辨識免疫球蛋白 E 的適體 (aptamer) 以硫金鍵結的方式固定在奈米金粒子上。接著在電極表面同時加上免疫球蛋白 E 與本實驗設計富含鳥嘌呤的免疫球蛋白 E 適體互補段 DNA，兩者以競爭反應的方式進行偵測。當免疫球蛋白 E 的濃度越高時，核酸適體抓取免疫球蛋白 E 之量也越高，使富含鳥嘌呤的免疫球蛋白 E 適體互補段DNA相對越少。接著利用亞甲基藍容易與鳥嘌呤產生特異性的吸附之特性，當電極表面之免疫球蛋白 E 適體互補段DNA越少時，電極表面亞甲基藍濃度越低，進一步使測得電化學電流訊號下降。反之，若免疫球蛋白 E 的濃度越低，富含鳥嘌呤的免疫球蛋白 E 適體互補段的修飾量也就越高，使亞甲基藍的訊號越大。而在此機制下設計的電極，針對分析物免疫球蛋白 E 的方波伏安法偵測極限為 0.16 pM (S/N = 3)，線性範圍則在 1 pM 到 0.1 μM之間 (R2=0.9958)。將牛血清白蛋白模擬人血清樣品，在 0.1 μM的牛血清白蛋白中測量五種不同濃度的免疫球蛋白 E 的回收率，低濃度 (10 pM) 免疫球蛋白 E 的回收率仍有 112.7% (RSD = 10.35%)。 本研究的結果相較於以往的文獻，選擇使用穩定性高、容易操作、高選擇性的適體與有良好電化學訊號的亞甲基藍結合，製造出具有高靈敏度、偵測範圍廣且不須前處理真實樣品的拋棄式電極。

Human immunoglobulin E (hIgE) is an important protein for its role in allergic disease. The extremely low concentration of hIgE in human serum makes it difficult to be detected. Therefore, a rapid, facile, sensitive and cost-effective detection method for hIgE becomes crucial and necessary. Aptamers are short DNA or RNA oligonucleotides that can bind to target molecules withhigh affinity and specificity. Due to their salient advantages, we use aptamer as the critical identification material for the detection method development of hIgE in electrochemistry. We used methylene blue (MB) as the electrochemical indicator due to its good redox characteristics and binding specifically to guanine bases in DNA sequence. Gold nanoparticles have good binding affinity with thiol group, and they can increase the surface loading of thiol-terminated capture DNA. We constructed a modified electrochemical sandwich model for IgE detection, using thiol-terminated capture DNA as the capturing probe, and complementary DNA sequence as the detection probe. The detection DNA, including part of G-rich bases in its sequence, can easily bind with MB to increase the redox signal and improve the sensitivity.In the presence of IgE, the amount of bounded report DNA will combine with capture DNA,which results in the decrease in the amount of report DNA on the sensing interface. A lower electrochemical signal attributed to the decrease amount of absorbed MB therefore, the redox current of the MB can reflect the concentration of the analyte. The proposed approach provides an alternative approach for sandwich protein assay using aptamers. This strategy successfully enhanced the sensitivity of biosensor and lower LOD to 0.16 pM. Wide linear range of 5 orders from 1 pM to 0.1 μM with R2 equal to 0.995 with RSD lower than 10.35%. The aptamer biosensor also has good selectivity and recovery. This method shows great potential in other protein analysis as well as disease diagnosis.