酵素熱生物感測器系統研究與應用

Research of Enzyme Thermometric Biosensor Detection System and Application

魏賢卿(Hsien-Chin Wei)

指導教授 ： 李允中

國立臺灣大學/生物資源暨農學院/生物產業機電工程學研究所/博士(2017年)

https://doi.org/10.6342/NTU201702384

摘要

『熱』是一生物體內酵素生化反應量及代謝量的重要指標。精密測量此一熱量變化，可以用來檢測基質濃度，也可以量化估算酵素活性及酵素動力學。
本研究旨在研發酵素熱生物感測器系統，並將其應用在生物介質的濃度及活性檢測。
本研究發展了三款酵素熱生物感測器系統，設計三種微熱卡反應槽，並分別應用於基質濃度測定、多通道微焦耳熱卡檢測及生鮮蔬菜酵素活性檢測等三個應用領域。
在基質濃度測定方面，本研究發展一種單通道簡易酵素熱生物感測器系統，用來直接快速檢測危害等級的過氧化氫殘留量，係結合醫療級負溫度係數熱敏電阻器做為熱感測器、壓克力(PMMA)反應槽、及自行組裝的高精確溫度檢測系統。經國家實驗室一級溫度校正，在20到30°C之間，本研究發展的溫度檢測系統之精確度達± 0.001 °C，線性相關係數為0.999。當以市售過氧化氫酶做為檢測用酵素時，過氧化氫殘留量線性檢測範圍約為31毫莫耳到1莫耳，判定係數R2為0.999，檢測極限約為53 ppm。
在多通道酵素熱生物感測器系統方面，量熱法生化測量提供各種優點，例如低浪費、低成本、低樣品消耗、短操作時間和省人力。多通道酵素熱生物感測器系統可以增強進行更高通量生物化學測量的可能性。焓傳感器陣列是多通道酵素熱生物感測器系統中的關鍵裝置。大多數的焓傳感器陣列使用惠斯頓電橋放大器來調節傳感器信號，但這種方法只適合用於零位檢測和低電阻傳感器。為了克服這些限制，本研究開發一種多通道同步微熱卡計測定(MCSA)平台、一種可調節微安培定電流源、一種使用微安培來激勵ES陣列電流環測量電路拓撲。MCSA平台包括測量單元，其包含多通道微熱卡計和自動同時注射器，以及信號處理單元，其包含多個ES信號調節器和數據處理器。這項研究著重於MCSA平台的建設;特別是測量的構造電路和熱量計陣列。平台的性能測試，包括電流穩定性、溫度敏感性、和熱敏性。傳感器響應時間和微熱卡計常數。平台檢測相對酶活性的能力也被證明。實驗結果顯示，本研究所提出的MCSA平台是一種靈活的強大的生化測量裝置，具有比現有替代品更高的通量。
在生鮮蔬菜酵素活性檢測方面，本研究開發了批式注射分析和酶–基質反應的酶活性檢測系統。設計多層空氣壁保溫微熱卡室和一次性聚合物反應容器加工的微熱卡裝置，用於通過實施單步測量一級反應的初始速度常數。溫度測量的靈敏度成功檢測為± 0.0015 °C。在反應容器中熱平衡後，通過計算初始速度和與標準酶活性的相關性，在4.5秒測定酶活性。當使用5歐姆微型鎳鉻線加熱器進行校準時，一次性聚合物反應容器的熱容量為6.654 J/℃，標準偏差為0.435 J/℃。可檢測的輸入能量約為10 mJ。當使用0.1 ml 100 mM 過氧化氫作為基質時，獲得來自牛肝的商業純化過氧化氫酶的一級反應的初始速度為0.0044℃/s/unit。其中獲得的小黃瓜、紅鳳菜、胡蘿蔔和甜椒的新鮮蔬菜汁具有一級反應的初始速度為0.0137、0.0118、0.0054和0.0021 ℃/s，換算未純化的過氧化氫酶活性約為31.3、26.9、10.5和4.8 units/ml。本研究的方法可以作為純化和未純化樣品中過氧化氫酶活性的快速且易於實施的測定。

關鍵字

生物微熱卡系統 ； 過氧化氫 ； 過氧化氫酶 ； 酵素活性 ； 生鮮蔬菜

並列摘要

"Heat" is an important indicator of the amount of biochemical reactions and metabolic activity of the enzyme. Precise measurement of this calorie change can be used to detect the concentration of the substrate, or to quantify
the estimated enzyme activity and enzyme kinetics.

The aim of this study was to study and develop the enzyme thermometric biosensor detection system and apply it to detect the concentration and activity of biological media. In this study, three type enzyme thermometric biosensor detection system were developed, and three micro-calorimetric reaction vessels were designed and used in three applications such as substrate concentration determination, multichannel micro-Joule calorimetric measurement and vegetables enzyme activity detection.

In the case of determination of substrate concentration. This articlebiosensor by a primary-grade thermometer calibration method, the accuracy of temperature measurement for the developed system was ± 0.001°C and the correlation coefficient R2 was 0.999 at temperatures between 20 and 30°C. The system response is linearity in the range between 31.25×10–3 and 1 M for detection of hydrogen peroxide, with a determination coefficient R2 of 0.999 and a detection limit of 53 ppm.

For multichannel enzyme thermometric biosensor detection system, calorimetric biochemical measurements offer various advantages such as low waste, low cost, low sample consumption, short operating time, and labor-savings. Multichannel microcalorimeters can enhance the possibility
of performing higher-throughput biochemical measurements. An enthalpy sensor (ES) array is a key device in multichannel microcalorimeters. Most ES arrays use Wheatstone bridge amplifiers to condition the sensor signals, but such an approach is only suitable for null detection and low resistance sensors. To overcome these limitations, a multichannel calorimetric simultaneous assay (MCSA) platform was developed. An adjustable microampere constant current (AMCC) source was designed for exciting the
ES array using a microampere current loop measurement circuit topology. The MCSA platform comprises a measurement unit, which contains a multichannel calorimeter and an automatic simultaneous injector, and a signal processing unit, which contains multiple ES signal conditioners and a
data processor. This study focused on the construction of the MCSA platform; in particular, construction of the measurement circuit and calorimeter array in a single block. The performance of the platform, including current stability, presents a micro-biocalorimetric system based on a thermistor and a disposable polymeric adiabatic calorimeter for measurement of hydrogen peroxide present at hazardous levels. After linearization of the calorimetric temperature sensitivity and heat sensitivity, was evaluated. The sensor response time and calorimeter constants were given. The capability of the platform to detect relative enzyme activity was also demonstrated. The experimental results show that the proposed MCSA is a flexible and powerful biochemical measurement device with higher throughput than existing alternatives.

For fresh vegetables catalase detection, an enzyme activity detection system based on batch injection analysis and enzyme-substrate reaction is developed. A precision calorimetric measurement device worked with a multi-walls thermal insulation chamber and a disposable polymer reaction vessel is used to measure the initial velocity constant of the first-order reaction by real time one-step procedure. The sensitivity of temperature measurement is successfully detected as ± 0.0015 °C. After thermal equilibrium in the reaction vessel, the enzyme activity is determined at 4.5 seconds by means of the calculation of initial velocity and correlation to activity of standard enzyme. The heat capacity of disposable polymer reaction vessel is found as 6.654 J/°C with a standard deviation of 0.435 J/°C when a 5 ohms miniature NIC80 wire (nickel 80% and chromium 20%) heater is used for calibration. The detectable input energy is obtained about 10 mJ. An initial velocity of the first-order reaction of a commercial purified catalase from bovine liver was obtained as 0.0044 °C/sec/unit when using 0.1 ml of 100 mM hydrogen peroxide as substrate. Where, fresh vegetable juice of cucumber, red cabbage, carrot and sweet pepper were obtained have an initial velocity of the first-order reaction as 0.0137, 0.0118, 0.0054 and 0.0021 °C/sec, respectively, the unpurified catalase activity are approximately 31.3, 26.9, 10.5 and 4.8 units per ml. This present method may serve as a rapid and easy-to-perform assay of catalase activity in purified and in unpurified samples.

並列關鍵字

Enzyme thermometric biosensor detection system ； Hydrogen peroxide ； Catalase ； Enzyme activity ； Fresh vegetables

參考文獻

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Cina, S. J., Downs, J. C. U. and S. E. Conradi. 1994. Hydrogen peroxide: a source of lethal oxygen embolism, Am. J. Forensic Med. Pathol. 15, 44-50.

Danielsson, B. and K. Mosbach. 1979. Determination of enzyme activities with the enzyme thermistor unit. FEBS LETTERS 101, 47-50.

European Commission, SCIENTIFIC COMMITTEE ON CONSUMER