壓電晶體免疫感測器於醫療檢驗之應用

Abstract

壓電晶體生物免疫感測器，其具有高靈敏性、高選擇性、即時分析、 成本低及快速的優點。在臨床檢驗上，極需利用這樣的特性以加速檢體的 測試，而給予醫師一個迅速確實的結果報告，達到患者病情治療的效果。 本研究將壓電晶體感測器結合免疫反應，利用polyethyleneimine及 glutaraldehyde固定抗體或抗原於壓電晶體上。而此法所製備之免疫探針 ，應用於以下三種檢測：（一）、幽門螺旋桿菌抗體的測定 萃取幽門 螺旋桿菌蛋白固定於壓電晶片，來偵測感染宿主血清中抗幽門螺旋桿菌抗 體的存在。實驗結果顯示於血清樣本高稀釋條件下，稀釋倍數與頻率衰減 呈線性相關。在此稀釋倍數(70-500倍)範圍內，不但可去除因溶液黏度性 質改變而致頻率變化的變因外，也降低了正常血清樣本非專一性結合的干 擾。此外增加注入樣本量至250μL，樣本於稀釋70倍下，感染血清與正常 血清其衰減頻率差距達100Hz。當測量完成時，晶片可以利用1M NaOH脫覆 晶體上的抗體，使晶片重複使用達10次以上。（二）、金黃色葡萄球菌的 鑑別 本實驗以Anti-protein A antibody，IgG或血漿固定於晶片做為 測量金黃色葡萄球菌探針，其中以Anti-protein A antibody探針效果最 好，於2.14 x 105/mL菌量下，即可因頻率衰減差20Hz而被偵測到，且不 受其它菌屬，例如大腸桿菌非專一性的干擾。將此探針應用於菌血症病人 血液篩選時，發現準確性不如預期理想，僅達50%，推測不同抗體來源所 製成之探針及固定化技巧的好壞是影響檢測結果的原因所在。（三）、心 臟藥物digoxin微量分析 以Anti-digoxin monoclonal antibody固定晶片 為探針，直接分析試液中digoxin的含量，測量極限只達mg/mL之範圍。為 提高測量靈敏度，改以BSA-digoxin conjugate 固定晶片為探針，利用加 入定量的anti-digoxin monoclonal antibody 與free digoxin，使BSA- digoxin conjugate 與free digoxin 競爭Anti-digoxin monoclonal antibody結合位置，以間接測定試液中 digoxin 的濃度。結果顯示，利 用競爭性間接測量時，測量digoxin濃度可低至1.04ng/mL，符合臨床偵測 血液中digoxin濃度之需求。 The advantages of piezoelectric crystal immunosensor are their high sensitivity, high selectivity, real-time analysis, low cost, and fast response. These properties are useful in a clinical diagnosis to accelerate the process of examination and to provide fast and accurate information to a doctor. In the present study, we use piezoelectric crystal immunosensor to develop three fast procedures to detect anti-Helicobacter pylori antibodies, Staphylococcus aureus and digoxin.1.Detection of anti-H. pylori antibodies Proteins extracted from H. pylori are immobilized on the crystal to detect the existence of anti-H. pylori antibodies in the host serum. The results revealed that when serum was diluted in a range of 1/70 ~ 1/500, the decrease frequency of the crystal was correlated to the concentration of serum. The decrease may be due to the liquid viscosity and the interference of nonspecific protein binding. The optimal condition is obtained when the serum was 70-fold dilution and the sample volume applied to the system was 250 mL. In this condition, H. pylori infected serum and normal serum result in a 100Hz difference of frequency decrease of the crystal. In addition, the crystal was regenerated after washing with 1 M NaOH.2. Detection of S. aureus in serumAnti-protein A antibody, IgG or human plasma are immobilized on the crystal for detecting S. aureus. Result showed that immobilized anti-protein A antibody was an appropriate probe for S. aureus detects. Frequency decrease was measured as low as bacterial density of 2.14 x 105 cells/mL. However, when blood culture sample from infected patients was appliedin this system, the specificity and sensitivity were low. We found that the anti-protein A antibodies from various commercial sources severely influenced the test accuracy.3. Analysis of digoxin concentrationBy using with immobilized anti-digoxin monoclonal antibody as a probe to detect digoxin concentration in sample did not provide a sufficient sensitivity. To overcome this issue, a competitive method with BSA-digoxin as a probe was used. By adding a certain amount of monoclonal anti-digoxin antibody to the unknown sample, we could indirectly measure the concentration of digoxin that compete by bound to immobilized BSA-digoxin. As low as 1.04 ng/mL of digoxin could be detected by this indirect method.