免標定電化學阻抗生醫感測器之微流體晶片開發

Abstract

隨著現代醫療科技發展的突飛猛進，人類的平均壽命跟著逐年增加，使得老年人口數量提升，導致高齡化社會來臨，因此，未來醫療照護的需求也將逐漸提升。也因此，醫療照護的觀念逐漸延伸到小型診所和一般家庭，因此有越來越多以定點照護(Point-of-care, POC)為訴求的生物感測器產生。由於目前心血管疾病是造成台灣國人死亡前三名的疾病，因此本研究希望能結合定點照護的概念，開發出應用於此疾病的生物感測器，檢測其相關的生物指標蛋白質(例如：C-reactive protein (CRP)與S100)。 為了達到定點照護的功能，小體積、低成本、易操作及足夠的靈敏度等特性，都是在設計生物感測器時必須考慮到的因素。由於光學式生物感測器有體積大、成本高與光路校正不易等問題，因此較不適合開發成定點照護的儀器；而電化學阻抗式生物感測器的靈敏度不差、校正方便，設計上也比較容易使體積微小化，因此本研究選擇後者做為生物晶片開發的基礎。為了降低成本、節省樣本使用量以及提升操作的方便性，我們將電化學的三極式電極設計成生物晶片，並結合微流道系統，形成微流體生物晶片。本研究利用Cysteamine與ATP作為晶片的連結分子，分別進行CRP與S100的抗體－抗原交互反應，並利用電化學阻抗分析法(EIS)，驗證微流體生物晶片之可行性。結果顯示，隨著抗原濃度增大，電子傳遞電阻的變化量(∆Ret)與其呈現相當線性之關係，可見生物晶片檢測之穩定性。整體的線性量測區間為10 ng/ml~10 μg/ml，檢測極限可達10 ng/ml，比美國心臟協會所公布的心血管疾病低危險群的1000 ng/ml(CRP)標準還要低於兩個濃度等級。此外，為了確保連結分子產生不變性以提升生物晶片修飾後的良率，我們希望能將新合成且穩定性較佳的導電連結分子(AS2SAc)與生物晶片做結合，因此利用螢光顯微術以及電化學阻抗分析法來驗證AS2SAc之鍵結能力與導電效果。結果顯示，AS2SAc確實有其鍵結效果；就EIS結果來看，Ret值約在幾千歐姆左右，屬於導電能力佳的連結分子。因此，若將微流體生物晶片與AS2SAc兩者結合，除了能保有手持式、可拋棄與小體積等優點，更增加了長時間保存的特性，未來將更適合應用於定點照護的生物感測器上。

With the rapid development of medical technology, average life span of human has greatly increased over the years. The trend of fast approach to an aging society for most developed countries has raised the demand for better medical cares. To improve the health care, it is vital to diagnose the diseases such as cancers and cardiovascular disease in an early stage. All of which have driven the concept of point-of-care testing (POCT) to small clinics and typical families. To achieve the above-mentioned vision, many portable biosensors are to be developed. Since cardiovascular disease (CVD) induced death remains on the top 3 death causes in Taiwan over the years, we hope to develop a biosensor for the detection of CVD biomarkers. The newly developed POCT utilized biomarkers such as CRP and S100 for diagnosis. Since typical optical biosensors have problems such as miniaturization difficulty, high cost and light alignment difficulty, etc., we adopted electrochemical methods for our biosensor development. A good biosensor developed for POCT implementation should have advantages such as small size, low cost, and ease of operation. Therefore, we miniaturized the three-electrodes onto a biochip, and combined it with microfluidics to construct a microfluidic biochip system. Here we used Cysteamine and ATP as our bio-linkers to perform the antibody-antigen interaction tests for the verifications of the microfluidic biochip’s feasibility. The proteins we used here are CRP and S100. The results showed that the change of electron transfer resistance grows linearly with the protein concentrations. The detection range was identified to be from 10 ng/ml to10 μg/ml, and the detection limit was 10ng/ml. This is two orders of magnitude lower than the concentration of a person faces a low risk of developing cardiovascular disease (AHA). Besides, we did the fluorescence test and the impedance measurement on a novel conductive linker, AS2SAc. This linker is stable and easy to preserve; therefore, we hope to improve the sensor stability by the use of the linker. From the experimental results, we found that the AS2SAc has a sufficient binding ability and a low Ret, which is suitable for the protein detection. Hence, we believe that by combining the microfluidic biochip and AS2SAc, the biosensor we developed can be very useful for POCT implementation.