多維度紙張微流體分析平臺應用於糖化血色素檢測

Abstract

本研究開發一多維度紙張微流體平臺結合免疫分析法與攜帶式Blue Red value 讀取儀，主要研究動機為達到定量分析血紅蛋白、糖化血色素以及疾病定點照護的目的。平臺整體由匣具與纖維素層析紙構成。匣具提供正向力，使得多層紙張能緊密配合，減少紙張層與層之間的空隙與降低外在干擾。紙張微流道以漸擴式圖案設計，讓試劑能夠均勻且穩定地在紙張微流道中流動，得到均勻、明確的檢測結果點。本平臺採用競爭型免疫分析反應為檢測機制，透過探針與目標蛋白競爭抗體位置，偵測檢體中糖化血色素與血紅蛋白的濃度。最後使用攜帶式Blue Red value 讀取儀，將檢測結果數據化，提供測試者判讀檢測結果，達到定量分析糖化血色素的目的。檢測過程，採用直流式免疫分析(Vertical Flow Test, VFT)，以直接滴入樣品於檢測區域進行檢測，試劑以垂直方向流動，有效降低檢測時所需樣品體積，縮減流體路徑，減少無效體積。並測試不同的吸收層紙張微流道圖案，進行紙張微流體平臺優化，目標降低檢測誤差與得到均勻的檢測結果點。本研究同步進行競爭型免疫分析法的試劑濃度探討，利用最佳化紙張微流體平臺，得到捕捉抗體、探針以及酵素的最佳化濃度。由分析結果證明血紅蛋白可獲得線性偵測極限為32 ~ 500 µg/mL、糖化血色素可獲得線性偵測極限為10 ~ 1000 µg/mL，僅需指針採血所獲得檢體量，即可進行檢測。檢測完成後，將檢測層紙張插入攜帶式Blue Red value 讀取儀中，進行檢測結果數據化，整個檢測在15分鐘內即可獲得分析結果。吾人期望此多維度紙張微流體平臺，能夠應用於糖尿病患者的病情監控與診斷以及一線醫療人員進行糖尿病的快速篩檢，達到即時疾病定點照護(Point of Care Testing, POCT)的目的。

We have developed a multi-dimensional paper platform combined with immunoassay and a portable Blue Red value reader in this study. The main motivation of this study is to achieve quantitative analysis of hemoglobin, glycated hemoglobin, and to achieve point of care testing. The platform is composed of housing and cellulose chromatography paper. The housing is processed with 3D printer and combined with paper during testing, so that multiple layers of paper contact more closely, reducing the gap between paper layers and removing external interference. In the testing, we use vertical flow immunoassay which can greatly reduce dead volume of reagents compared with lateral flow systems. The paper microfluidic channel is designed with a gradual pattern, so that the reagent can flow evenly and stably in the paper microfluidic channel, and a uniform and clear detection result point can be obtained. The molecules that are not attached to the detection area flow to the absorbent layer by capillary force. In this way, the detection area can reduce detection errors and non-specific attachments. The platform was used to complete a competitive immunoassay to quantitatively analyze of hemoglobin and glycated hemoglobin. The results show that the detection range of hemoglobin is 32 to 500 µg/ml, and the detection range of glycated hemoglobin is 10 to 1000 µg/ml. The detection can be performed only by pointer blood sampling. After the test completed, the detection layer is inserted into the portable Blue Red value reader, and test results are digitized to achieve the purpose of (Point of Care Testing, POCT). We hope this multi-dimensional paper platform can be applied to the monitoring and diagnosis of diabetic patients and the rapid screening of diabetes by first-line medical staff.