Analysis of Amperometric Response to Cholesterol according to Enzyme-Immobilization Methods

Cholesterol is the precursor of various steroid hormones, bile acid, and vitamin D with functions related to regulation of membrane permeability and fluidity. However, the presence of excess blood cholesterol may lead to arteriosclerosis and hypertension. Moreover, dietary cholesterol may affect blood cholesterol levels. Generally, cholesterol determination is performed by spectrophotometric or chromatographic methods, but these methods are very time consuming and costly, and require complicated pretreatment. Thus, the development of a rapid and simple analysis method for measuring cholesterol concentration in food is needed. Multi-walled carbon nanotube (MWCNT) was functionalized to MWCNT-$NH_2$ via MWCNT-COOH to have high sensitivity to $H_2O_2$. The fabricated MWCNT-$NH_2$ was attached to a glassy carbon electrode (GCE), after which Prussian blue (PB) was coated onto MWCNT-$NH_2$/GCE. MWCNT-$NH_2$/PB/GCE was used as a working electrode. An Ag/AgCl electrode and Pt wire were used as a reference electrode and counter electrode, respectively. The sensitivity of the modified working electrode was determined based on the amount of current according to the concentration of $H_2O_2$. The response increased with an increase of $H_2O_2$ concentration in the range of 0.5~500 ${\mu}M$ ($r^2$=0.96) with a detection limit of 0.1 ${\mu}M$. Cholesterol oxidase was immobilized to aminopropyl glass beads, CNBr-activated sepharose, Na-alginate, and toyopearl beads. The immobilized enzyme reactors with aminopropyl glass beads and CNBr-activated sepharose showed linearity in the range of 1~100 ${\mu}M$ cholesterol. Na-alginate and toyopearl beads showed linearity in the range of 5~50 and 1~50 ${\mu}M$ cholesterol, respectively. The detection limit of all immobilized enzyme reactors was 1 ${\mu}M$. These enzyme reactors showed high sensitivity; especially, the enzyme reactors with CNBr-activated sepharose and Na-alginate indicated high coupling efficiency and sensitivity. Therefore, both of the enzyme reactors are more suitable for a cholesterol biosensor system.

효소고정화 방법에 따른 콜레스테롤 검출용 바이오센서의 전류 감응도 분석

콜레스테롤의 신속하고 정확한 새로운 분석방법을 모색하기 위하여 본 연구에서는 전기적 전도성이 우수한 MWCNT를 이용하여 전극을 제작하였고, 여러 가지 효소고정화 방법을 통해 전기화학적 감응도 분석을 실시하였다. MWCNT의 전도성을 향상시키기 위해 아민기를 도입한 MWCNT-$NH_2$를 제조하였고, MWCNT-$NH_2$/GCE에 PB를 점착하여 작업전극을 제조하였다. 제조한 작업전극은 0.5~500 ${\mu}M$ $H_2O_2$ 농도 범위에서 농도가 증가함에 따라 전류가 비례적으로 증가하였고, 검출한계는 0.1 ${\mu}M$로 나타나 전극이 높은 감도를 가지고 있음을 확인하였다. 또한 콜레스테롤 검출을 위해 적합한 효소 반응기를 제작하기 위해 담체인 aminopropyl glass beads, CNBr-activated sepharose, Na-alginate, toyopearl beads에 cholesterol oxidase를 고정화시켜 바이오센서의 콜레스테롤 표준용액에 대한 감응도를 측정한 결과, aminopropyl glass beads과 CNBr-activated sepharose는 1~100 ${\mu}M$ 범위에서 선형관계를 보였으며, Na-alginate는 5~50 ${\mu}M$의 범위에서, toyopearl beads는 1~50 ${\mu}M$ 범위에서 선형관계를 나타내었다. 검출한계는 제작된 효소반응기 모두 1 ${\mu}M$로 나타나 콜레스테롤에 대한 높은 검출력을 보여주었으나, 특히 CNBr-activated sepharose와 Na-alginate를 이용한 효소반응기가 높은 coupling efficiency와 감응도를 보여 콜레스테롤 검출을 위한 본 바이오 센서 시스템에 적합한 것으로 나타났다.