多頻道人體免疫抗體石英壓電感測器的研製與應用

摘要

本研究旨在研製一多頻道的固定化碳六十/免疫球蛋白抗體石英壓電晶體感測器，並運用此固定化的石英壓電晶體感測器偵測溶液中的免疫球蛋白(IgG, IgA, IgM)，再利用碳六十/PVC做為壓電感測器的塗佈膜，探討碳六十與人體免疫球蛋白抗體(Anti-IgG, Anti-IgA, Anti-IgM)間的作用力。研究結果顯示碳六十能和抗人體免疫球蛋白結合，呈現不可逆的化學性吸附現象。此碳六十－抗體可由碳六十與人體免疫球蛋白抗體反應得到，並以FT-IR光譜做鑑定。並探討碳六十/PVC塗佈量以及人體免疫球蛋白抗體的濃度對免疫球蛋白吸附量的影響，發現塗佈4μg碳六十/PVC以及注入0.6mg/mL的 Anti-IgG、0.3mg/mL的 Anti-IgA、0.014mg/mL的 Anti-IgM可獲得最大訊號。
將固定化的碳六十－人體免疫球蛋白抗體石英壓電晶片接上自組的電子線路界面，偵測溶液中的免疫球蛋白，可得到頻率下降的訊號變化，且為可逆的物理性吸附，證實碳六十與抗人體免疫球蛋白結合之後仍具有活性，且頻率訊號與人體免疫球蛋白濃度呈一線性關係，其偵測靈敏度分別為9.62×103Hz/(mg/mL)、1.86×105Hz/(mg/mL)、4.48×105Hz/(mg/mL)，偵測下限為9.97×10-4mg/mL、3.19×10-5mg/mL、1.72×10-5mg/mL。此免疫感測器可重複偵測免疫球蛋白達五次以上，有不錯再現性。
利用此現象更深入探討人體免疫球蛋白抗體－免疫球蛋白反應的條件，結果顯示其最適pH值在6～7之間，最適溫度25～350C。若將此碳六十－人體免疫球蛋白抗體石英壓電晶片保存於40C下，可保存其有效性達六天以上。
本研究亦探討當人體中常見的生化物質，如glucose、uric acid、cystein、tyrosine等存在時對免疫球蛋白感測的影響，結果顯示其選擇係數皆達90％以上。

Multi-channel Piezoelectric Immunosensor for Human Immunoglobulins
Abstract

Fullerene C60-anti-human immunoglobulin, C60-anti-human IgG, C60-anti-human IgA and C60-anti-human IgM, coated piezoelectric crystals were prepared and applied in a multichannel piezoelectric quartz crystal immunosensors for human IgG, human IgA and human IgM, respevtively. The immobilization of anti-IgG, anti-IgA and anti-igM onto fullerene C60 were studied with a C60-coated piezoelectric crystal detection system. The partially irreversible responses for anti-human immunoglobulin were observed by the desorption study, which implied that anti-human immunoglobulin could be adsorbed on C60 by chemisorption.
The immobilized C60/anti-human immunoglobulin coating materials were successfully prepared and identified with FTIR spectrometry. The fullerene C60-coated piezoelectric quartz crystal sensor was employed to study the interacton between C60 and anti-hunan immunoglobulin. In order to obtain maximum adsorption of anti-human immunoglobulin on C60-coated crystal , the C60-anti-human immunoglobulin coated quartz crystal were prepared with 4μg C60 crystal coating and 0.6mg/mL Anti-IgG, 0.3mg/mL Anti-IgA, 0.014mg/mL Anti-IgM in water.
The C60-anti-human immunoglobulin coated PZ quartz crystal immunosensors with homemade computer interfaces for signal acquisition and data processing were developed and applied for detection of human immunoglobulin,respectively. The C60-anti-human immunoglobulin coated PZ immunosensors for IgG, IgA and IgM exhibited linear frequency responses to the concentrations of human IgG, IgA and IgM with sensitivities of 9.62×103, 1.86×105 and 4.48×105 Hz/ (mg/mL). And the C60-anti-human immunoglobulin coated PZ immunosensors showed detection limit of 9.97×10-4, 3.19×10-5 and 1.72×10-5mg/mL for IgG, IgA and IgM in water.
The reproducibility and lifetime of these immobilized C60-anti- human immunoglobulin coated PZ immunosensors were also investigated and discussed.
Effects of pH and temperature on the frequency response of the immunosensor were also investigated. Optimum pH of the solution and optimum temperature for the sensor were observed at pH=6~7 and 25～350C. The interference of various common species in the human blood, e.g. glucose, uric acid, cystein and tyrosine showed nearly no interference to the detection of human IgG, IgA and IgM with the piezoelectric immunosensor.

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