| Summary: | 博士 === 國立成功大學 === 醫學工程研究所 === 89 === This study utilizes piezoelectric quartz crystal (PQC) sensors as an analytical tool for biomedical applications. Application fields contain blood coagulation tests, developing an affinity biosensor and interfacial study by integrating an electrochemically analytical technology. This investigation aims to extend the applications of PQC sensors to clinical tests, and to developing novel biosensors and basic interfacial sensor research. Topics covered herein include blood coagulation studies and the development of an affinity biosensor. The contents of each chapter are listed below:
Part 1: Determination of plasma prothrombin time using a PQC sensor.
The extent to which a PQC sensor was influenced by liquid viscosity was studied herein, as was the sensor’s application to estimating blood coagulation time and its application to estimating blood coagulation time. The relationship between the frequency shift (Df) of PQC and the physical properties of the contacting liquid was also examined. Glycerol solutions with various weight percentages were adjusted to various viscosities. The Df is inversely linearly proportionate to (rlhl)1/2 in general blood conditions (0.14 s-1/2g-1cm2.). The sensing function to liquid density and viscosity was utilized to monitor blood coagulation and determine coagulation time in real time.
PQC sensors with adequate sensitivity to slight viscosity changes are employed to monitor blood coagulation. The case of an anticoagulated plasma specimen, prothrombin time (PT) was determined by the PQC sensor based on the introduction of tissue thromboplastin (TF) and calcium ion. The PQC data reveals that the best linear relation ship in a double-logarithmic plot of PT versus TF concentration in the range of 5.466 ~ 22.311 units/ml appeared at 90% of the total frequency shift. The PQC result was compared with a commercial optical coagulometry and showed a strong linear correlation (ca. 0.98). The PQC sensor can potentially be used in basic hematology research owing to its real time monitoring capability, which overcomes the drawbacks of the end-point method used in traditional coagulometers. Furthermore, the PQC sensor has an advantage in satisfying future demand for bedside and home-care products in that only a 20 ml sample is required for testing.
Part 2: Determining heparin levels in blood with activated partial thromboplastin time using a PQC sensor.
A PQC sensor was used to determine both whole blood activated partial thromboplastin time (WBaPTT) and plasma activated partial thromboplastin time (PLaPTT) induced by anticoagulant heparin. The PQC sensor results revealed a linear relationship between WBaPTT (or PLaPTT) and heparin levels in clinically relevant concentrations (0~0.4 unit/ml). The mean of individual R2 (= 0.9491) for a regressive curve between WBaPTT and heparin concentrations was shown sufficiently clearly. The PQC method can be employed to assess the influence of heparin through determining WBaPTT, since its sensitivity (P< 0.01) is comparable to that of aPTT by optical coagulometry (OCaPTT). Furthermore, the results of WBaPTT with various heparin concentrations (n = 9) were found to be closely correlated with those of OCaPTT (correlation coefficient = 0.9441). Linear calibration plots were extended into 3 units/ml of heparin in PLaPTT and WBaPTT.
Measured results indicate that the prototype coagulometer based on PQC sensor has a closer linear relationship than the optical coagulometer in high-dose ranges of heparin. It has been suggested that the PQC method is more convenient, which may be useful in clinical situations for rapid monitoring heparin therapy using a small volume (20 ml) of whole blood specimens. The PQC method has three advantages in heparin assay: rapid analysis (from 80 to 600 sec), wide detection range (0~3.0 units/ml) and convenient sample preparation (whole blood is available).
Part 3: Studies of whole blood coagulation using PQC sensors.
This section builds on the previous two chapters regarding applications of PQC sensors to hematological studies, and particularly in assessing the practicability of applying PQC sensors to whole blood coagulation tests. Long-term (3000 sec) and real time viscosity monitoring was conducted during whole blood clotting. Viscosity is insignificant in PQC responses between whole blood and plasma. A step-like response curve is also obtained from analyzing whole blood coagulation within 1000 seconds, an acceptable time period, in clinical trials. A comparison of responses between whole blood and plasma coagulation does not reveal interferences on the surface of the PQC sensor by cells and proteins in blood. Additionally, it is practical to apply the PQC sensor to studies of whole blood coagulation via research on whole blood recalcification time.
A PQC sensor was used to determine whole blood clotting time (CT) to quantitatively analyze heparin concentrations in blood. An obvious linear relationship existed between whole blood CT and heparin concentration from 0 to 0.1 units/ml, but the examination took over 1000 seconds. Consequently, while the results are analytically useful, the approach not be practical for application in clinics.
Whole blood activated partial thromboplastin time (aPTT) was determined using a PQC sensor to investigate high concentrations of heparin in-vitro and ex-vivo. Ten thousand units of heparin were injected into each subject undergoing heparin treatment following a cardio catheter. Plasma aPTT could not be determined in over half of the blood sample using a commercially optical coagulometer. PQC sensors conducted all of the tests and the test results reveal an appropriate linear relationship, indicating the effectiveness of PQC in discriminating between anticoagulation and a normal condition. The “receiver operating characteristic” plot was used to comparatively assess sensitivity and specificity between different coagulation analyzers. The analytical results revealed that PQC performs better in distinguishing heparin anticoagulation than an optical coagulometer.
Part 4: Development of a heparin sensor based on PQC.
This investigation attempted to develop a heparin sensor for clinical use, possessing directive assessment, easy operability and a wide calibration range. Absolute concentrations of heparin in phosphate buffer solution (PBS, pH 7.4) were determined using PQC as an affinity biosensor, and electrochemical impedance spectroscopy (EIS) was used to investigate immobilization of protamine and heparin assay. Constructing a heparin sensor requires using protamine as a specifically recognized elements and using simple physical adsorption as an immobilization method to develop a heparin sensitive surface on a PQC gold electrode.
The effectiveness of physical adsorption in immobilizing protamine was confirmed by examining the preparation condition, including incubation time and protamine concentration. The reduction in oscillating frequency of PQC (ca. -100 Hz) was maximized when applying 20 mg/ml protamine in PBS with a 20 minute incubation period. Heparin adsorption onto protamine-modified electrode in PBS revealed an exponential-like binding curve, and a long time was required to reach the steady state in the PQC frequency response. Furthermore, judging from the initial slope (df/dt) and frequency change (Df) of PQC two linear calibration curves were obtained after a binding interval (600 seconds) for heparin concentrations from 0 to 3.0 and 7.0 units/ml, respectively.
Ten thousand ppm of bovine serum albumin (BSA) was used as mimic plasma to assess the interference of proteins in blood with a protamine-modified sensory surface. Interference causing a —50 Hz frequency shift will influence the sensing function of the heparin sensor, but might not be lethal. The EIS system was corporately employed to investigate preparation procedures and the sensing function of heparin sensors. In EIS analysis, calibration curves with a linear concentration range of 0~3.0 units/ml were also obtained for heparin in PBS when ferrocyanide was used as an electroactive marker. The EIS method will be conducive to designing micro sensing arrays that allow the development of inexpensive biosensors.
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