Research Of Aptamer-based Piezoelectric Quartz Crystal Biosensor

Objective:To optimize and improve the performance of home-made piezoelectric quartz crystal biosensor and detecting analysis software, and to choose optimal method for aptamer immobilization on the surface of gold membrane. Then an aptamer-based piezoelectric sensor assay was established to detect human immunoglobulin E and acquire the parameters about this kind of biosensor.Methods:1. Fine processing technique was used to fabricate single quartz crystal wafer (13×13mm) and to plate the gold electrode of 4.0mm in diameter on the two sides of quartz crystal. The single sensor was composed of the detecting cell and AT-cut quartz crystal with a fundamental frequency of 10MHz .Then sensors were arrayed to construct 2×5 plug-play type array. Detecting software applied to record and analyze frequency changes was updated from PESA-2.0 to PESA-4.0. The new software was designed based on Visual C++, which fixed some known issues and added many useful functions for laboratory research and clinical detection. Later, the stability of system was tested in gas phase and liquid phase individually.2. Avidin was immobilized on the gold surface of piezoelectric quartz crystal sensor by self-assembly of 3,3'-dithiopropionic acid (DDPA). The process of avidin immobilization was showed by the changes of frequency on the screen. Then the changes of frequency were compared with different concentrations of avidin and different pH value. Finally, the data were analyzed by one-factor analysis of variance via SPSS software.3. Anti-IgE aptamers were immobilized on the gold surface of piezoelectric quartz crystal sensor with thiol method and biotin-avidin method respectively. The reaction between anti-IgE aptamer and IgE was monitored in time on piezoelectric sensor. The frequency shifts were compared in reactions with different concentrations of IgE. The data were analyzed by independent-samples T test via SPSS software.4. Anti-IgE aptamers were immobilized on the gold surface with biotin-avidin method. Different concentrations of IgE were detected with this sensor and the calibration curve of IgE was drawn. The detection limit could be calculated based on the calibration curve and three times standard deviation of signals of PBS blanks. Non-specific response signals of BSA, IgG and IgE were monitored to show the detecting specificity of aptamer-based sensor. We tried to regenerate the sensor surface after binding IgE by rinsing the aptamer-coated chips with HCL, NaOH, EDTA, urea and formamide individually. Aptamer-coated chips were stored in PBS with 0.1% sodium azide and the stored chips were used to detect IgE in 35 days.Results:1. AT-cut quartz crystal had strong vibration ability and stable frequency in liquid phase. In ten minutes, the changes of frequency were less than±1Hz in gas phase and±2Hz in liquid phase respectively.2. In the sensor array, the plugging and playing of any sensor had no effect on other sensors which were in detection. All of the sensors had stable frequencies in multichannel detection. The stability of system was good enough for this research.3. The PESA-4.0 software designed mainly for clinical detection had added some useful functions such as recording the information of patients, recalling history records, curve fitting, and calibrating. Moreover, the functions of image editing and automatic updating of frequency were helpful for laboratory research.4. With one-factor analysis of variance, the frequency changes in PBS (pH6.2) were not statistically different from those in PBS at pH5.8 and pH6.6, but there were significantly higher frequency shifts in PBS (pH6.2) than those in PBS when pH>7.0. And the 0.2 mg/ml solution of avidin was most appropriate for avidin immobilization.5. The chips coated by the two methods were used to detect IgE. Results showed that thiol method was unfit for aptamer immobilization. For thiol method, the reacting tendency of IgE was not so obvious and the reproducibility of detection was unsatisfactory. However, biotin-avidin method gained higher frequency changes in the reactions of IgE , lower limit of detection and wider liner range than thiol method.6. Anti-IgE aptamers were immobilized to detect IgE with biotin-avidin method. For the aptamer-based sensor, the lowest limit of detection was 0.055mg/L and the liner range was 0.1-2.5mg/L. Non-specific signals of this sensor were less than 5% of specific signals of IgE at the same concentration. Of the five reagents, EDTA was the best reagent for regeneration of aptamer-coated chips and the sensor could retain 91.5% of the original detecting signals after five regeneration cycles. Moreover, the aptamer-coated chips could be stored in the binding buffer (0.1%NaN3) for 21 days without obvious loss of activity.Conclusions:1. The piezoelectric sensor array we constructed had strong vibration ability in liquid phase and had stable frequency in multichannel detection. So this system could be used to detect target molecules by immobilizing all kinds of recognizing molecules including aptamers. The PESA-4.0 software designed originally for clinical detection was also helpful for laboratory research.2. 0.2 mg/ml solution of avidin at pH6.2 was selected as the optimal during avidin immobilization on gold surface of piezoelectric quartz crystal sensor.3. The thiol method which was proved effective for probe immobilization on gene sensor was obviously unfit for aptamer immobilization. However, avidin-biotin method is a good way to immobilize aptamers on the gold electric surface of the quartz crystal.4. The aptamer-based piezoelectric sensor could detect 5.5 ng IgE and the non-specific signals were very low. So this kind of aptamer-based sensor was hopeful to be applied to clinical laboratory diagnosis.5. Compared with antibody-based piezoelectric sensor, aptamer-based sensor had lower cost, more regeneration cycles and longer time for the storage of chips. This series of experiments showed the superiority of aptamer detection.