Experimental Study Of Piezoelectric Gene Sensor By PNA And The System Of Biology Magnified Of "RecA Protein-complementary Single Strand DNA" Probe

Objectives:Our study focuses on the sensitivity of detection system of quartz oscillation gene sensor (QOGS) array with probe of bis-PNA and "RecA protein-complementary single strand DNA", and explores the feasibility of clinical samples test by such system.Methods:1. Technology of fine processing was firstly used to make the solo QOGS and then the gold electrode was etched around two sides of sensor. Different thickness and diameter of electrodes were compared in order to determine its influence to the detection of gene hybridization. Based on it, plug-plucked style 2×5 QOGS array was constructed. We also developed the self-oscillating circuit and the PESA version 2.0 software applying to record and analyze frequency. So the detection system of QOGS array was constructed by combing all these components.2. To investigate the superiority of the gene hybridization of the piezoelectric sensor by comparing the probe of PNA with the ordinary probe of DNA. The probe of PNA and DNA were immobilized on the gold surface with biotin-avidin method, and hybridized with completed target sequence, one mismatched and two mismatched target sequences respectively. Frequency shift and the time consuming by the reaction of the two different probes were finally observed.3. Bis-PNA probe for detecting HBV was immobilized on the gold electrode surface of QOGS array firstly. To improve the sensitivity of piezoelectric gene sensor by increasing its surface load, "RecA protein-complementary single strand DNA" probe was designed to combine bis-PNA / dsDNA complex.4. On the late stage of experiment, Bis-PNA probe was used to directly detect the extracted genomic DNA from 36 positive clinical samples caught hepatitis and 14 negative sera samples.Results:1. Quartz crystal with AT cutting style was selected to be elementary plate of sensor because of its benefit for stable oscillation in liquid phase. The thickness of gold electrode between 50nm to lOOnm would be most suitable for detection of gene hybridization, while the diameter 4.0mm would be less insert loss and can afford more quality burden. Many advantages such as convenience, economy would be possessed with plug-plucked style QOGS array which was constructed based on solo QOGS.2. Self-oscillating circuit that was developed by ourselves had strong vibromotive ability in liquid phase and it's stabilize of frequency could also meet the request of experiment. The new adjustable plug-plucked sensor was capable of solving the problem that the frequency of quartz crystal was difficult to be stable in liquid phase. The frequency of the sensor is capable of reaching ± lHz in gas and liquid phase.3. Self-developed PESA version 2.0 software applyed to record and analyze frequency had many functions such as setting up primary parameters, recording change of frequency in real-time, plotting figure of frequency change automatically, and so on. Data would be transferred to computer automatically after the collection and analysis.4. The capability of recognizing mismatched base by PNA probe was higher and the time consuming of hybridization was shorter than that of DNA probe. PNA had highly specificity to combine with the DNA target sequence. And so it would improve the detection specificity of piezoelectric gene sensor by using PNA probe.5.1 The effects of different concentrations of RecA protein as 0.5, 0.85, 1.5, 3, 4.5, 6mg/ml were observe. Results showed frequency change increased firstly and then decreased when the concentration of "RecA protein-complementary single strand DNA" probe varied from 0.5 to 3mg/ml. When the concentration was 3mg/ml,the change of the frequency was most obvious.5.2 When the concentration of the RecA protein was 3mg/ml, frequency changed differently when adding different concentrations of ATP Y S . Frequency change increased obviously when the concentration of ATP Y S varied from 5 to 12.5 u M. And then the frequency decreased when the concentrations of ATP Y S were higher than 12.5 U M.6. The effect of mass-frequency was more evident, and hence the sensitivity of piezoelectric gene sensor array is improved significantly by using "RecA protein-complementary single strand DNA" probe. 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 gene sensor.2. Special optimization was processed by self-oscillating circuit to QOGS array, when it oscillated in liquid phase, and hence realized the stable oscillation in liquid phase.3. PESA version2.0 applying software was a kind of new-style software, which can record and analyze the resonance frequency of QOGS array at real time.4. The new adjustable plug-plucked style sensor can increase the stability of genesensor in liquid phase.5.1 PNA probe can hybridization with target nucleic acid sequence with higher specificity than that of the capability of recognozing ordinary DNA probe. Matched and mismatched base by PNA probe was higher,while the tolerant degree of PNA hybridized with complementary target DNA sequence was lower than that of DNA / DNA. So, choosing PNA as the probe may improve the detection specificity of piezoelectric gene sensor.5.2 HBV genomic DNA from clinical samples could be detected directly by designed Bis-PNA probe. And the range of HBV DNA was located in the detecting linear range of QOGS array exactly. And hence a good technical platform was successfully constructed with the detection system of QOGS array when it applied to detect HBV quantitatively in clinical samples. Sensitivity and specificity of QOGS showed no significant difference when it compared with quantitative PCR, but less time was consumed and no probe should be marked at QOGS.6. The sensitivity of piezoelectric gene sensor array is improved significantly by using "RecA protein-complementary single strand DNA" probe.