Experimental Studies On Detection Of Urinary Micro Protein By Piezoelectric Quartz Crystal Immunosensor Array

Objectives:To construct plug-play type micro-array immunosensor and apply it to clinical laboratory, and to optimize possible factors affecting the efficiency of antigen-antibody reaction in solution.An end-point judging method of QCM was established and discussed. A novel detection platform of piezoelectric quartz crystal oscillation frequency was established in order to detect the clinic samples.Methods:1. Fine processing technique was used to make single quartz crystal wafer and then the gold electrode around two sides of the sensor was plated. Different thickness and diameters of the gold electrodes were compared in order to determine their impact on the detection of immunosensor. On these basis, a 2 × 5 plug-play type micro-array immunosensor was constructed. The old frequency acquisition card was replaced with new style one. The operating system was updated to those support windows NT. The composite of temperature controlling system was added. Double power management system was applied. Optimized Self-oscillating TTL circuit and self-made PESA version 2.0A software were applied to record and analyze frequency changes. Then the universal detection platform for oscillation frequency of piezoelectric quartz crystal micro-array sensor was constructed.2. Monoclone anti-microprotein antibody was immobilized on the gold electrode surface of piezoelectric quartz crystal immunosensor by SPA. A series of experiments were firstly conducted to find the concordance of Ab immobilization and the best antibody concentration in immobilization. The temperature-time relationship during the Ab-Ag reaction was discussed. The influence of PBS pH value and PBS ionic concentration on the frequency shifts was explored. On this base, a new fitting method to decide the end-point was established and then standardized3. We designed and fabricated piezoelectric quartz crystal Micro Protein micro-array immunosensor to detect a -1-MG, 3-2-MG, MA and IgG in urine. The sensitivity, detection limit, nonspecific response and working area of piezoelectric quartz crystal immunoglobulin micro-array immunosensor were observed. At last, clinical samples were detected and the results were compared with those detected by conventional immunoassay in order to explore the feasibility of the detection of clinical samples.Results:1. 10MHz AT-cut quartz crystal with gold-coated electrodes of 4.0mm in diameter and 100nm~~150nm in thickness was found to be of strong vibration ability and stable oscillation in liquid phase. They also had high sensitivity and good quality loading, which would be most suitable for the detection of immune reaction.2. The micro-array immunosensor had met the requirements on the aspects such as single sensor and immunosensor array system, the temperature controlling system, PESA V2.0A software and the double electric power.3. The new adjustable plug-play sensor was capable of solving the problem as the frequency of quartz crystal was difficult to be stable in liquid phase. The frequency of the sensor was capable of keeping an ±1 Hz in gas and ±2Hz in liquid phase.4. The immobilization of antibody by SPA was easy to operate and need less time. The amount consistency and biological reaction activity of the antibody were good. It was a good way of antibody immobilization by SPA.5. Under the optimal temperature, the piezoelectric quartz crystal immunoglobulin sensor micro-array could complete the detection in 20 min with the same sensitivity, specificity and detection limit as conventional immunoassay. The results were compared with that of conventional immunoassay, and the correlation coefficients were good, which showed that the micro-array immunosensor could be used for clinical detection.Conclusions:1. 10MHz AT-cut quartz crystal with gold-coated electrodes of 4.0mm in diameter and 100nm~~150nm in thickness is most suitable for the micro-array immunosensor.2. The new adjustable plug-play type sensor is capable of solving the problem as the frequency of quartz crystal is difficult to be stable in liquid phase.3. The new type quartz crystal immunosensor has many advantages, such as stable anduser friendly which can be used to detect other immune reactions.4. SPA is a good way to immobilize antibody on the gold electric surface of the quartz crystal.5. Low exponent function is a good model to fit curve and we can easily find the end-point by finding the minimum slope rate. This is a feasible method to decide the end of the reaction in the immunosensor.6. Piezoelectric quartz crystal micro-array immunosensor is a new method for detecting combined units of Micro Protein in urine, which is hopeful to be applied to clinical laboratory diagnosis widely.