Construction Of A Novel Piezoelectric Biosensor Based On Protein-DNA Reaction

Objective1.To improve the performance of piezoelectric quartz crystal biosensor detection system made by us.2.To confirm the specific reaction between NF-κB cis-acting element(DNA) probe and the specific protein NF-κB p65 subunit and the nucleoprotein,by the EMSA method.3.To discuss the influencing factors of the novel protein-DNA piezoelectric biosensor and optimize the reaction condition.Methods:1.Fine processing technique was adopted to fabricate single quartz crystal wafer(AT cut 13×13mm) with a fundamental frequency of 10MHz and to put the gold electrode of 4.0mm in diameter on the both two sides of quartz crystal.The single sensor was arranged to construct 2×5 insert-plug model 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++,and added many useful functions suitable for laboratory research and clinical detection.In addition,the stability of system was tested in gas and liquid phase respectively.2.The cis-acting element probe of NF-κB modified by thiol group were immobilized on the gold membrane surface of the quartz crystal sensor by thiol-self-assembly technique, and was hybridized with the specific protein NF-κB p65 subunit.When some parameters, such as probe concentration,pH value and ion concentration,were changed,the corresponding changes of the frequency of each sensor were recorded and analyzed through the computer simultaneouly.The reaction condition was optimized by SPSS statistic software.3.The combination specific between NF-κB cis-acting element(DNA) probe and the specific protein NF-κB was confirmed with EMSA method. 4.The different concentrations of p65 were used as standard substance to draw detection calibration curve of p65 and determinate the level of the nucleoprotein.In addition,the detection specifity of the biosensor was probed by different sequence thiol group probe immobilized on the surface of piezoelectric crystal.Results:1.AT-cut quartz crystal has strong vibration ability and stable frequency both in gas and liquid phase.In ten minutes,the changes of frequency were less than±1Hz in gas phase and±2Hz in liquid phase respectively.2.The software of PESA- 4.0 which is designed for clinical detection mainly has been added some useful functions such as recording the information of patients,recalling history records,curve fitting,and calibrating,etc.Moreover,the functions of image editing and automatic updating of frequency were helpful for laboratory research.3.In the EMSA electrophoresis results,a lag electrophoresis band could be observed, which probe marked by the isotope combined with NF-κB p65 subunit and the nucleoprotein union resulted in the lag electrophoresis band,compared with the blank control group.But the binding band could not be observed in the blank control group.This phenomenon demonstrated that the probe and the protein combined specificity.4.The p65 subunit was detected by using the piezoelectric quartz crystal sensor.The optimized reaction condition was 2.0μmol/L probe concentration,pH 7.5,50mmol/L MgCl₂ and 15min for reaction time,respectively.5.Piezoelectric Biosensors different concentration p65 was detected in the 10-40ng/ul concentration of p65 concentration and frequency decreased linearly related,Non-specific signals of this sensor were less than 5%of specific signals of p65 at the same concentration.Conclusions:1.The hardware of piezoelectic quartz crystal sensor detection system was designed perfect relatively.It works stably and the software of the system used easily and stably.The studies imply that the detection system can be used for detection and research.In addition the PESA-4.0 software designed originally for clinical detection was also helpful for laboratory research.2.The piezoelectric examination has obtained the good result and the examination time is 15 min.The non-specificity signal is small,which indicates the specificity of sensor is ideal and the selectivity is high.All the data demonstrated that this platform has the good application prospect in the trans-acting-factor examination.3.The EMSA electrophoresis experiment implied that the specific probe designed in our experiment have the specific combination both with NF-κB p65 standard protein and the nucleoprotein.It was indicated that this sensor detection system is a good examination specificity.4.This sensor detection system has more advantages compares with traditional EMSA method.For example,it doesn't need isotope and the reaction time is shorter.Furthermore, it can online observe and record the corresponding data with high flux.And it provide a new technical platform for the eukaryotic gene expression regulation study.