Construction Of BisPNA Leaky Surface Acoustic Wave Gene Sensor Detection System

Objectives:1. To improve the leaky surface acoustic wave (LSAW) gene detection sensor system based on the previous study.2. To design a bis-peptide nucleic acid (bisPNA) probe, combined with a high sensitivity and precise LSAW sensor, a new type of bisPNA -LSAW gene sensor detection system was built.3. To explore the factors that influence bisPNA probe hybridization with target sequence and to optimize the reaction conditions.Methods:1. Precise machining was used to make the LSAW biosensor with a dual resonance style based on the dual delay linear LSAW sensor developed previously. A reflexed bar array was added on both sides of the LSAW sensor and this new type of LSAW sensor was constructed.2. The resonance circuit and software for data collection and analysis were improved and a temperature controlling system was added to construct a new type of LSAW biosensor detection system.3. DNA probes for the HPV target sequence were immobilized on the surface of the new LSAW sensor. Following optimal reaction conditions developed previously, the complemental target sequence was added and the phase change resulting from by the hybridization reaction was observed and compared with previous findings.4. The key factors, including pH value, ionic concentration and probe concentration, were explored and optimized with the new bisPNA-LSAW gene sensor detection system. And then the sensitivity and linear range of detection was confirmed.Results:1. The signal reflex distance between import and export interdigital transducer was increased by introducing the reflexed bar. Conscquently, the phase change caused by target hybridization to the DNA probe was improved significantly.2. The detection efficiency and precision was also improved by progress in the data collection and analysis software. The core program of the software was optimized and upgraded, hence it runs more stable and processes faster. And introduction of the temperature controlling system provided an optimal temperature environment for biological reaction.3. Phase changes caused by the hybridization reaction of the bisPNA and the target sequence varied significantly at different pH values. First the phase change increased and then it decreased when pH values of the hybridization buffer was varied from 5.8 to 8.0. And the response signal was highest at pH6.6 and 6.8 while the equilibrium time was significantly different (P<0.01), and a shorter time was needed, at pH6.6. In addition the equilibrium time increased suddenly when moving from pH6.6 to pH8.0.4. Phase change increased first and then decreased when ion concentrations were varied from 10mmol/L to 100mmol/L. and 20mmol/L was the watershed. And the equilibrium time for hybridization increased when ion concentrations was increased, and it changed significantly among different groups.5. When probe concentration was varied from 0.001μmol/L to 4.0μmol/L, the phase change increased first and then it decreased when hybridized with the HPV target DNA, and 1.0μmol/L was the watershed. Change in equilibrium time were not obvious.6. Phase change was first enhanced and then it shift gently with hybridization HPV target DNA when the concentration of target was varied from 1pg/L to 1mg/L, and 100μg/L was the saturation point. The statistic linear regression equation was△P =2.3392 lgC +14.584 among a range of target concentrations from 1pg/L to 100μg/L and the correlation coefficient was 0.9622. No evident change in tendency was observed regarding the equilibrium time for hybridization.Conclusions:1. The LSAW biosensor with dual resonance was more suitable for detecting molecular biology experiments in liquid phase compared with the dual delay linear LSAW sensor previously developed. The introduction of a reflexed bar array increased the signal noise ratio and hence increased the sensitivity of the sensor. 2. The improved LSAW sensor detection system attained optimal experimental conditions for every component including oscillation circuit, temperature controlling system and the software.3. For smooth hybridization of bisPNA and HPV dsDNA, it was crucial to have a slightly acid environment, i.e pH6.6. The hypothesis of"Hoogstein-strand first"during the procedure of bisPNA reacting with dsDNA was confirmed by our results.4. Ion concentration was found to be another important influence factor. Low ion concentration was beneficial not only to improve the signal of bisPNA and dsDNA, but also to improve the hybridization velocity of bisPNA and dsDNA. While equilibrium time was reduced significantly by high ion concentration.5. Optimal immobilized probe concentration was 1.0μmol/L. The phase change caused by the hybridization reaction was first increased and then shifted gently, that is to say, like a typical saturation curve, in accordance with the increase of the target concentration.6. The detection sensitivity of the LSAW sensor can reach 1pg/L. It improves greatly compared with the bulk acoustic wave gene sensor.