利用振動式自感測壓阻式微懸臂梁監測人體血漿中內部路徑凝血反應

Abstract

隨著現代人的生活以及飲食習慣的改變，造成心血管疾病患者數量逐年遞增，而為了預防及治療由血液與血管壁異常作用造成的血管栓塞，病患須依賴抗凝血劑的治療。然而，如果使用了過量或不足的抗凝血劑量，則會造成大量出血或者無法成功預防治療等後果，因此使用抗凝血劑的病患需定時且即時地監測血液的凝固狀態是否在正常範圍內。因為目前血液的檢驗屬於醫療等級，病患若需監測血液凝固狀態，需要接受醫療院所的生醫檢測流程，然而醫療院所的檢測流程從檢體的處理、運送、儀器排程、上機測試到最後取得報告的時間相當冗長，並無法確實地達到即時監測的目的，因此本研究使用微奈米機電技術開發出能夠快速檢測的振動式自感測壓阻式微懸臂梁凝血感測器，搭配快速傅立葉轉換演算法分析訊號，應用於抗凝血劑肝素(heparin)用藥監測的評估方式—部分活化凝血酶原時間(APTT)的量測。 量測方式參考分析血液凝固狀況的機械式Sonoclot分析儀，本研究使用定頻率、定振幅的制動器驅動壓阻式微懸臂梁，使其在待測樣品中振動，利用血凝樣品年度變化時微懸臂梁受力情形也會產生變化作為監測基礎，擷取感測器之壓阻訊號以推知代測樣品的黏度性質變化。搭配快速傅立葉轉換演算法，可得知在致動器振動頻率的振幅值，本研究以此振幅值來反映微懸臂梁的受力情形，進而分析並得到部分活化凝血酶原時間。 首先利用不同濃度的甘油水溶液進行實驗來了解微懸臂梁感測器在不同黏度下的受力情況，實驗結果可看出微懸臂梁阻值改變量的10Hz振幅與黏度有正相關的趨勢，且線性度良好。同時也將微懸臂梁感測器之訊號與Reynolds number以對數相關的分析方式來表現，得到log(∆R/R_0 )=0.7922[-log(Re) ]-0.0314方程式來表示本研究所使用的微懸臂梁在液體中振動的表現(R2=0.929)證實本研究所開發的振動式自感測壓阻式微懸臂梁能分辨不同黏度的液體。接著將量測標的改為血凝樣品的實際凝固情形，利用自行設計的快速傅立葉轉換演算法來處理訊號，可得知在血凝樣品發生凝固過程中特定頻率的振幅變化情形，以振幅明顯驟升所需時間作為微懸臂梁所量測到的部分活化凝血酶原時間(Son-aPTT)，三重複實驗結果得到第一級血凝品管液的Son-aPTT為23.2秒(標準差為1.25秒)；第二級血凝品管液的Son-aPTT為47.5秒(標準差4.55秒)；第三級血凝品管液的Son-aPTT為83.5秒(標準差4.32秒)。將微懸臂梁感測器與商用儀器的量測數據進行統計學上判斷差異性之T檢定，發現在95%信賴區間內兩者並無差異，且都在藥品的參考部分活化凝血酶原時間範圍內，證實本研究的確能用微懸臂梁感測器監測到凝血反應發生時纖維蛋白生成的情形。 本研究開發之振動式自感測壓阻式微懸臂梁感測器屬於微奈米機電技術，有可微型化與降低成本之潛力，而且後端訊號處理可與訊號擷取系統一同設計在軟體中，無論是在醫療院所的床邊照護或者定點照護領域中，均有很大的發展空間。

In this study, we successfully developed a real-time, miniaturized coagulation monitoring sensor by using an externally vibrated, self-sensing piezoresistive microcantilever for disposable point-of care coagulation device. Patients with cardiovascular disease to avoid the thrombosis formation are required anticoagulant by continuous infusion. The infusion of anticoagulant during the previous 24 hours need to be allocated to have their therapy monitored by activated partial thromboplastin time (aPTT, an index for blood coagulation status). With the increasing use of anticoagulant, the need of point-of care coagulation devices has become necessary. Activated partial thromboplastin time (APTT) is a measure of intrinsic pathway and common pathway of blood coagulation, and it is an index for anticoagulant therapy to determine the blood condition in blood coagulation reaction. In this study, the measurement was performed by vibrating the piezoresistive microcantilever immersed in the sample liquid at a fixed frequency of 10Hz and fixed amplitude of 40μm with an external vibrator. The acquired of resistance change in microcantilever processed by Fast Fourier Transform algorithm, and the resistance at amplitude 10Hz indicates the amount of force exerting to the cantilever. During the coagulation reaction, the viscosity of the sample would suddenly rise due to the clot formation, and the increased force can be sensed when the resistance amplitude in 10Hz rises. Activated partial thromboplastin time can be obtained by the time needed for fibrin clot formation. Because of the method was initiated by Sonoclot analysis, so we called the measurement result as Sonoclot signature-like activated partial thromboplastin time (Son-aPTT). The amplitude of resistance in the specific frequency performed well linear correlation in calibration with absolute viscosity changes of glycerol/water solutions(R2>0.99), and we also found out that the amplitude-absolute viscosity curve behave differently in very low absolute viscosity, probably due to the decrease in viscous drag for low absolute viscosity. Three types of commercially standard samples for coagulation activated partial thromboplastin time measurements were used for the testing of microcantilever sensor. The measured results of resistance amplitude in specific frequency with specific patterns of signature indicated the viscoelastic changes during blood coagulation reaction. In coagulation control level 1, the Son-aPTT measured by microcantilever is 23.2 sec with std. of 1.25 sec; Son-aPTT = 47.5 sec with std. of 4.55 sec in coagulation control level 2; and Son-aPTT = 83.5 sec with std. of 4.32 sec in coagulation control level 3. Compare with commercial coagulation device, the aPPT measured show no difference between microcantilever sensor and commercial device in 95% confident level, and all lie in the reference aPTT ranges. The experiment results support that the aPTT measured by microcantilever sensor perform well and precise. Moreover, the patterns of resistance amplitude can be further analyzed for another useful information of blood such as MA for fibrinogen level in TEG analysis. This microcantilever sensor has performed well in real-time measurement for point-of-care coagulation monitoring, after miniaturized and optimized, its potential can be well worth looking forward to.