壓阻式微懸臂梁生物感測器之製作與C-反應蛋白偵測之研究

Abstract

疾病問題一直是人類重要課題之ㄧ，尤以生物分子間鍵結情形為基礎之檢測技術更是佔有一席之地，透過不同生物指標可以準確檢測出可能發生之疾病以達到及時診斷之目的，其中C-反應蛋白( C-reaction protein，簡稱CRP )在近幾年應用相當廣泛，在急診臨床的診斷或是心血管疾病風險的評估，都有相當重要的發展。隨著奈米生物技術日趨成熟，微生物感測器近幾年逐漸取代佔有空間大、所需成本過高的傳統檢測系統，成為量測C-反應蛋白的最佳利器。 而目前市面上微生物感測器大多仍需使用化學或是螢光標記之方式，為此本研究選用以力學為基礎的微懸壁梁式生物感測器做為檢測機制，且為了避免傳統光學式微懸臂梁感測器需架設複雜光場及容易受到環境影響所造成的檢測誤差，而選用壓阻式微懸臂樑生物感測器(Piezoresistive micro-cantilever biosensor)進行實驗。本研究利用生物固定化的技術將抗體固定在微懸臂量上，並透過專一性使相對應的抗原與抗體結合，抗原抗體間鍵結時由於表面應力改變造成懸臂梁彎曲，此時懸臂梁中之壓阻阻值也會隨之改變，因此在實驗架構上配合惠司通電橋作為轉換機制，將抗原抗體結合反應透過壓阻效應轉換為電壓訊號，經由後端電路放大器等處理將訊號傳出到電腦分析。本研究使用C-反應蛋白作為生物指標，量測不同C-反應蛋白抗原濃度下造成訊號之差異，並探討不同溫度下壓阻式微懸臂梁生物感測器反應區內溫度之變化及溫度對於壓阻式微懸臂梁的輸出訊號影響，以其能使壓阻式微懸臂梁生物感測器系統準確率達到最佳化。

C-reaction protein( CRP ) is becoming one of important biomarkers in cardiovascular disease, myocardial infarction( MI ), and atherosclerosis. Higher concentration of CRP in serum elevates the risk of those diseases. Despite of existing techniques of surface plasmon resonance( SPR ), enzyme-linked immunosorbent assay( ELISA ) and others developed, the present piezoresistive microcantilever biosensors are sensitive, potentially inexpensive, label-free and of ease-of-use in miniaturization. To be detected, the microcantilevers are placed inside a microfluidic system that has a volume of several ten microliters. As probed biomolecules are transported onto a sensing surface of microcantilevers, specific recognition occurs, resulting in biomolecular conformation change and associated nanomechanic deflection of induced surface stresses. The deflection is mostly detected by an optical lever of detection system. Despite sensitive detection, the entire optical measurement is bulky and hardly aligned in readout system. For biomolecular recognition, the piezoresistive cantilever is required for operation in a phosphate buffered saline solution or human plasma which is to maintain a specific pH environment of a strong electrolyte. As a result of device electrical insulation to its solution, the piezoresistive cantilever is required to be surrounded by the insulating material to prevent electric leakage and unwanted chemical reaction on the sensing surface. The selected silicon nitride is used to conform to the requirements for the insulting material. The cantilevers are designed in 150 or 200 μm long, 50 μm wide and about 1.4 μm thick. The integrated piezoresistive resistors are 100 μm long, 50 μm wide and 200 nm thick, and the expected resistance is approximately 4 kΩ. Meanwhile, the gold layer (30 nm thick) coated on the cantilever surface is necessary for chemical reaction of biolinker, and thus the sensing surface of specific protein adsorption and its associated recognition. Preliminary result of CRP detection was obtained by this piezoresistive microcantilever. The device is confirmed to be tested in solution environment and thus its preliminary feasibility. More testing is required for further verification in CRP detection.