| ObjectivesBased on the construction of piezoelectric immunosensor micro-array detection system and optimization of the experimental conditions, a new method was established to detect Pseudomonas aeruginosa (PA) and identify its serotype. And the possibility of clinical application was also explored.Methods1. The fine processing techniques were firstly used to make the solo quartz crystal and then the gold electrode was etched around two sides of sensor. Based on it, clamped 2.5 style and plug-play 2.5 style immunosensor micro-array were constructed successively. Also we developed the self-oscillating circuit and the PESA version 2.0 software which were applied to record and analyze frequency changes. So the detection system of immunosensor micro-array was constructed by combing all these components.2. PA monoclonal antibody was immobilized on the gold electrode surface of immunosensor micro-array by SPA method and Biotin-Avdin method, respectively. Advantages and disadvantages of two methods were compared specifically including the amount of immobilized antibody, the response characteristics, detection limit, nonspecific response and so on.3. Regeneration ability of immunosensor micro-array was compared by four methods including glycine-HCl buffer, NaOH+HCl, citrate buffer and piranha detergent.4. To find the optimal temperature condition, the influence of temperature was investigated based on the amount of immobilized antibody, the response characteristics and the stored condition in different temperatures.5. Monoclonal antibody for detecting PA was immobilized on the gold electrode surface of immunosensor micro-array. And the curve was statistically drawn up about the relationship between the concentration of PA and decrease of frequency.6. Compared with the method of slide agglutination, Grouping Antisera of PA were immobilized on the gold electrode surface for the serological identification of PA.7. To explore the possibility of the clinical application, 50 clinical samples were detected and identified by our immunosensor micro-array, the results were compared those with conventional experiments.Results1. The piezoelectric immunosensor micro-array could meet the request of our experiments.1.1 The sensitivity and responsibility of gold-coated piezoelectrode achieved great advance after the optimization.1.2 Clamped style and plug-play style immunosensor micro-array, successfully realized the stable oscillation in gas and liquid phase (?IHz in gas and ? Hz in liquid phase).1.3 Self-developed PESA version 2.0 software which were applied to record and analyze frequency had many functions such as setting up primary parameters, recording frequency change at real-time, plotting figure of frequency change automatically, and so on. Data would be transferred to computer automatically after the date collection and analysis.2. The sensitivity of two antibody-coated methods was comparative, but the SPA method is easy to operate, the amount of immobilized antibody and biological response characteristics are better than that with biotin-avidin method, meanwhile, the nonspecific response and the time reaching stable were decreased.3. The cleaning efficiency and the reproducibility of piranha detergent would be much better than that of other detergents. The frequency shifts of response only decrease 5% even after reused 30 times.4. Temperature played quit important role in the immunosensor micro-array detection system.4.1 The amount of immobilized antibody reached maximum at 4;4.2 The frequency shifts reached maximum and it need shortest equilibrium time when temperature varied from 35 to 37.4.3 The antibody-modified crystal stored at 4'C has much longer lifetime than that stored at room temperature, and only caused 30% loss of the original activity after 60 days.5. The statistic exponential regression equation was AF =12.6625 XC01757 among the range of PA concentration from 103 to 109 cells/ml and the correlating coefficient was 0.9949. When unknown concentrat...
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