和尚蟹幾丁質皮膜於生物感測的應用

Abstract

將台灣種短趾和尚蟹(Mictyris brevidactylus Stimpson, 1858)施予1 M鹽酸處理去除蟹殼上的碳酸鈣等灰質(1 hr)，接著施予1 M氫氧化鈉處理分解蟹肉蛋白質後(2 hrs)，可由其背部甲殼取得顏色接近透明、天然的幾丁質皮膜。皮膜厚度約為20 um至50 um，含水率約為90%至98%，分子量在1000 kDa以上。透過機械性質測試發現和尚蟹幾丁質皮膜擁有的拉力強度(5.57 MPa)甚至優於厚度為120 um之Parafilm○R (4.39 MPa)，皮膜細微孔徑構造能成功阻隔待測溶液中如蛋白質高分子與帶電干擾物影響(例如：維生素C)，藉由循環伏安法與電化學阻抗分析結果得知，和尚蟹皮膜本身不具有電化學活性，亦能在不影響中性待測物質之質傳與擴散的前提下，有效地排除血清蛋白質非特異性吸附，保護電極表面。 本研究欲利用和尚蟹幾丁質皮膜作為酵素固定化擔體物質，以架橋劑戊二醛(glutaraldehyde)固定化葡萄糖氧化酵素(glucose oxidase, EC 1.1.3.4)，架構高靈敏度葡萄糖電極與流動注射式葡萄糖生物感測器，並應用於檢測市售乳酸醱酵飲料之葡萄糖含量。另外也以相同手法固定化膽鹼氧化酵素(choline oxidase, EC 1.1.3.17)，組裝流動注射式膽鹼感測器，應用於篩選抗乙醯膽鹼酯酉每活性之天然物質(可作為抗阿茲海默症天然藥物)以及評估血清膽鹼酯酉每之活性(肝臟功能的一項重要指標)。實驗結果發現以和尚蟹酵素皮膜所組裝的生物感測器，其耐用度皆可達到兩個月以上，並且顯示出良好的感測器性能，包括葡萄糖或膽鹼之檢出上限、檢測極限、重複性、再現性等。

Chitinous membranes from the dorsal part of Taiwanese soldier crabs (Mictyris brevidactylus Stimpson, 1858) were purified by a sequence of acid/base treatments (treated with 1 M HCl for 1 hr, and then with 1 M NaOH for 2 hrs). The thicknesses of the clear chitinous membranes were measured to be 20 um - 50 um, the moistures were 90% - 98%, and the molecular weights were estimated over than 1000 kDa. The natural membranes showed higher tensile strengths (5.57 MPa) than the laboratory-use thin film, Parafilm○R (4.39 MPa, 120 um in thickness). Moreover, the fine pore-sized structure of the membranes could effectively impede the diffusion of problematic biomacromolecule and charged interferents such as ascorbate through the membrane. Both the cyclic voltammetric curves and the electrochemical impedance curves revealed that the biomembrane was electrochemical inert, and the electrostatic interaction between positively charged amino groups on the surface of chitinous membrane and negatively charged human serum albumin particles could extensively reduce the electrode fouling by non-specific adsorption of serum proteins. Our study utilized the natural chitinous membrane as the supporting material for enzyme immobilization by a cross-linking reagent, glutaraldehyde. For glucose sensing, glucose oxidase (EC 1.1.3.4) was immobilized to construct a glucose sensing electrode, and a flow injection glucose biosensor. These two systems were successfully applied to evaluate the glucose content in commercial soft drinks and fermentation broths. For choline sensing, choline oxidase (EC 1.1.3.17) was immobilized to construct a flow injection choline biosensor, this system was proved to be useful in screening natural substances with acetyl cholinesterase inhibitory activities that may become potential neutraceuticals for preventing Alzheimer’s disease, and was also used to estimate serum cholinesterase activity, a useful clinical index for liver function. All the experimental results demonstrated the constructed biosensors were sensitive, reproducible, and durable to glucose or choline biosensing for at least two months.