Experimental Study And Clinical Application Of Detecting SNP With Piezoelectric Biosensor

BackgroundsRecent years, piezoelectric biosensor is becoming a focus in biomolecule detection field. The basic principle is that the characters of its oscillation are highly responded with the changes of the surface mass adsorption. It could detect many biomolecular real-timely, sensitively and specifically, such as DNA, antibody, enzyme, bacterium, based on different recognition elements. In recently research, it had been used in many research fields such as molecular recognition, clinical diagnosis, molecular filming, surface property research, poison research and so forth. In addition, piezoelectric biosensor is easy to control and shape minimized,so it is flexible to be used in clinical diagnose.A SNP represents an alternate nucleotide in a given and defined genetic location at a frequency exceeding 1 % in a given population. As the mostly major in the human genetics variability, SNPs have immense potentiality in the study of disease'etiopathogenisis, risk assessment, prevention and treatment. It is now one of the main areas in the post-genomics era. These recently results confirmed that not only directly connected to disease, SNPs is also useful in the prediction of drug reaction and risk assessment in particular sensitivity popular. For these reasons, SNP detection technology is very important and can be the new area of the clinical diagnosis technology research.Although many SNP sites have been found to be linked with some disease, but lacking of simply, effective and cheap SNP detection technology make it still a toy in laboratory. For the advantage of piezoelectric biosensor, it is useful to use it in SNP detection.In SNPs detection, only one nucleotide variation occurred in the DNA sequence, the detection methods need to detect the one nucleotide mismatch target sequence from samples. The mass shift is very little and account for a big challenge to piezoelectric biosensor. It requires more sensitive and stable biosensor. In this study, we firstly improved the stability of piezoelectric biosensor and then confirmed that biotin-SA-HRP system can be used to amplify the signal of piezoelectric biosensor and improve its detection sensitivity. Based on that, we constructed a high sensitive Piezoelectric SNP biosensor. And then, we used EIS to evaluate it and optimize the reaction conditions. Finally, we use this system to detect one HBV related SNP site in the clinical samples, and studied its concordance with the RFLP method.Methods and Results1. Use enzyme signal amplification system to establish a high sensitivity Piezoelectric SNP biosensora) The feasibility study on the use of biotin-SA-HRP signal amplification system in piezoelectric biosensor: The biotin labeled or unlabeled probe was separately immobilized on the piezoelectric crystal wafer. The SA-HRP was added to bind biotin, and then the DAB substrate was added, the frequency shift was record and analyzed. The results showed that comparing with the unlabeled probe, biotin labeled probe had a remarkable frequency shift, with it, the biosensor can detect as low as 10-13mol/l ssDNA. It suggested that after binding biotin, SA-HRP catalyzed DAB and formed insoluble precipitation, and give a big mass shift on the piezoelectric crystal wafer. These results suggested that the Biotin-SA-HRP system can be used to amplify the piezoelectric biosensor signal, and improve its sensitivity.b) Use Biotin-SA-HRP system and SBE technology to establish a high sensitivity piezoelectric SNP biosensor: two target sequences with only one nucleotide (A/G) difference was added and hybridized with unlabeled probe which immobilized on the crystal wafer. Three DNA polymerase (Taq, Pfu and Klenow) were added separately to extend one base on the sequence of probe with biotin-dUTP. And then the SA-HRP and DAB was added, the frequency shift was recorded and analyzed. The results showed that the A-containing sequence brought a more remarkable frequency shift than G-containing sequence. The system can discernment these two sequences based on their different signal shift, and the pfu polymerase is more suitable in this system. The results also showed that this method detection limit is 2×10-11mol/l,and compared with normal method, it is more stable and more quickly, the signal detection time can reduced to 20min.2. To establish electrochemical impedance system and evaluate piezoelectric SNP biosensor: The electrochemical impedance system we established has three parts, piezoelectric element, three electrode system and electrochemical workstation. To characterize impedance shift of thin Au film, we set some setting suitable parameters and detect the impedance when biomaterial modification on the Au film with probe and target DNA. Results suggested that abnormal impedance of blank film could influence the stable detection. And we optimized the piezoelectric detection system by characterizing the Au film impedance with EIS. According to the Au film area we used, the best probe immobilized concentration is 2μmol/l and the suitable immobilized and hybridized duration is 60 min. we also detected impedance after every step of piezoelectric SNP detection, and found that it happened correspond impedance shift to biological reaction.3. Use piezoelectric SNP biosensor to detect clinical blood samples: According to the sequence of T392C, we designed the specific probe and primers. 44 clinical blood samples were collected and DNA genomes were extracted as PCR template. The result suggested that piezoelectric SNP biosensor could detect the SNP specifically. Compare with RFLP, the detection rate of TT is 94.7%, and the rate of CC is 87.5%. Two methods have no variability.Conclusions1. Enzyme signal amplification system could detect target DNA specifically and amplify the piezoelectric signal significantly. It has great versatility and can be used in different types of piezoelectric biosensors in detecting tiny mass.2. EIS is a powerful tool to provide information of various biochemical events occurring in biosensors and is useful in establishing and optimizing piezoelectric detection system and protocol.3. The piezoelectric SNP detection system we established can be used to clinical detect single nucleotide polymorphism from blood sample. And we also have designed a protocol on detecting ESR1 T392C SNP site with our system, and studied its features of frequency shift. Compare with RFLP, piezoelectric SNP detection system is more flexible to be used in clinical SNP detection.