以紙作為基底之疾病及酵素動力學檢測系統

Abstract

以紙為作為檢測平台的基底，發展臨床監護診斷的技術，在近年大量蓬勃發展。由於紙具有低成本、輕薄可攜、種類眾多和方便改質及操作的特性，常被應用在建立新的生化試驗。在本研究中，我們以特製的噴墨技術，將蠟印在濾紙上形成類似一般檢測用96孔盤的輪廓，透過加熱形成疏水性的邊界，以建立一多重檢測的平台。在第一部分的實驗中，我們將HRP酵素與血管內皮細胞生長因子之單株抗體結合，並在紙孔盤上完成酵素呈色反應並定量抗體，之後利用抗體抗原的專一性結合嘗試在紙上操作酵素連結免疫分析法(paper-based ELISA)。我們嘗試透過空氣動力驅動的微流道系統，將此抗體包覆於海藻膠微米球體內，再用紙的孔盤來檢測其包覆效率。在第二部分的實驗中，我們研究酵素在紙的纖維上反應的機制，也就是一個非勻相的情形。同時我們也期望可以達成一些目前比較常見的一些健康檢測，首先我們嘗試操作葡萄糖跟蛋白質的檢測，並進一步嘗試天冬氨酸氨基轉移酶(AST)以及鹼性磷酸脢(ALP)的檢測。透過拍照以及影像處理軟體，我們完成上述四種反應的定量。在酵素動力學的部分，我們觀察到代表酵素阻力的Michaelis 常數在非勻相(紙的纖維)的情況下，在HRP以及葡萄糖的組別有60%的增加，而在鹼性磷酸脢的組別並沒有太大的影響。結果顯示酵素呈色反應可在紙上操作並顯示出肉眼可辨別的結果，並且此結果可透過影像處理來量化達到檢測的目的。我們相信這樣的平台可提供開發中國家一個新穎的疾病篩檢策略，若透過進一步的改造，也可廣泛應用於自我健康管理、食物品質控制以及環境監控。

Paper-based sensors are rapidly immerging to meet the needs for point-of-care diagnose. Due to its low cost, portable, widely available and easy to operate properties, it has attracted lots of attention in biochemical analysis field. In this work, paper 96 well plate was fabricated via wax printing method to build up a multiple diagnostic system. horseradish peroxidase (HRP enzyme) was conjugated with monoclonal antibody to vascular endothelial grow factor (VEGF antibody), and the quantification of the enzyme was carried out on paper 96 well plate. The influence of temperature and conjugated antibody was also investigated. To test the feasibility using this system for drug screening, HRP-linked VEGF antibody was encapsulated in alginate microspheres via an air pressure driven nozzle and used as a model drug. And the encapsulation efficiency of VEGF antibody was reveal by this paper system. Also, through the recognition of VEGF antibody toward VEGF protein, we perform paper-based ELISA to prove the immuno bioactivity of this system. Another part in our study is to investigate the enzymatic reaction on paper fiber, which is an inhomogeneous condition. Meanwhile, we want to accomplish some common diagnostic trials on paper system. Firstly, glucose and bovine serum albumin (BSA protein) assay was carried out on paper 96 well plate. Afterwards, aspartate transaminase (AST) and alkaline phosphatase (ALP) assay was carried out with different immobilization order. Quantitative analysis was achieved by photographing and processing by imaging software. The Michaelis constant for paper plate and plastic plate was calculated by measuring the initial reaction rate of each group. We observed a 60% increase in Michaelis constant for HRP and glucose oxidase group and no significant difference in ALP group. The quantification of four colorimetric reactions can be successfully carried out on this paper plate system. The result acquired can be distinguished by naked eye and digitalized by imaging software. In addition, the cost for a single trial was highly reduced. With these preliminary data, we believe this paper based system can provide a novel alternative for diagnose purpose in developing countries. And with further modifications, this paper-based system can have a wide potential application in long tern health management, food quality control and environment monitoring.