| BackgroundThe completion of Human Genome Project has revealed that genetic diseases and cancers are closely associated with the mutation in the sequence of particular genes. Among these mutations, single nucleotide polymorphism (SNP) is the most abundant genetic variation and it occurs once every 100–300 bases. Therefore, it is particularly important for researches of pathogenesis and early therapy of corresponding human diseases if we can achieve early, accurate, simple, rapid identification of the SNP. Up to now, many methods for SNP analysis have been reported. However, these traditional methods are not widely available, mainly because of time-consuming, complex operation and many factors. Although there are some new equipments and technologies including DNA microarray and SNP detector, these have only been applied to large-scale scientific research institutions because of high cost. Accordingly, to explore simple, inexpensive, accurate and easy to be clinically popularized detection methods for SNP is still of considerable interest.DNA piezoelectric biosensors have been widely used for the assay of the structure for nucleic acid analysis and detection of single base mutation due to its easy operation, rapid response, high mass sensitivity and low cost. At the same time, Leaky Surface Acoustic Wave (LSAW) sensor has been developed as a new bio-diagnostic techniques, combining with physics, materials, microelectronics, signal processing, technology in many other areas of science. The using of piezoelectric crystals of special shear to the incentive LSAW, when small changes occurred on the surface of the mass loaded, LSAW transmission frequency and phase change accordingly, which can be applied to analyse biological reaction accured on the bio-sensitive membrane. Comparing with the traditional body-wave sensors, LSAW sensor is of higher sensitivity, higher accuracy, better repeatability and reliability, lower cost. Therefore, LSAW sensor has a very broad application prospects.Based on preliminary work in our laboratory, this study constructed a new LSAW-SNP gene sensor detection approach by combining the usage of resonator-based LSAW sensors and the DNA ligase and enzyme biological signal amplification system. To take Japanese encephalitis virus E gene as a target SNP site, a preliminary study of LSAW-SNP gene sensor detection in samples of the actual application was investigated.Methods1. Based on preliminary studies, the construction of two-terminal resonant-type sensor, the phase-recording software, the temperature control system was improved. A LSAW sensor system more suitable for liquid detection was constructed. The software and hardware system were improved with NI data acquisition virtual instrument system. In addition, RFID sensor was designed.2. A variety of factors of the DNA-LSAW gene sensor detection system for liquid sensors detection were explored and optimized. Moreover, the time of the hybridization reaction was determined.3. The feasibility study on the use of enzyme biological signal amplification system in LSAW gene sensor was explored. The biotin labeled and unlabeled probes were immobilized on the LSAW gene sensor wafer respectively, and then HRP-avidin was added to bind biotin, and finally DAB/ H2O2 substrate was added, phase changes were recorded and analyzed.4. Enzymes biological signal amplification system and Taq DNA ligase reaction were combined to establish a LSAW-SNP gene sensor detection system. Two oligonucleotides as two target sequences with only one nucleotide (A/G) difference were synthesized, respectively. 5' thiol-modified DNA probes of Probe-1, through self-assembled immobilized at the surface of gold electrode, allele of the variable base is located in the probe 3'end with target-G the variable form of the corresponding. Add the target sequence and the target sequence with SNP site downstream fragment completely complementary biotin-labeled probes of the Probe-2, after hybridization the thermal stability Taq DNA ligase connected DNA incision, and then denatured by the alkali solution with target sequences fully complementary probe retained the sensor surface. Then HRP-avidin was added, and finally the DAB and H2O2 were added, insoluble precipitate was formed on the gold surface, resulting in increased phase LSAW sensor gene, thereby nucleotide variation to the information site was detected successfully.5. LSAW-SNP gene sensor detected real samples. A SNP site of Japanese encephalitis virus E gene (A2293G) was taken for the target P3 strain and SA14-14-2 attenuated strain of the RNA were extracted, then for RT-PCR amplification. The ssDNA PCR products were purified for sequencing. 1nmol/L ssDNA PCR product was detect with the established LSAW-SNP gene sensor method.Results1. Two-terminal resonant-type LSAW sensor vibrated stably both in gas and liquid phase. In ten minutes, the changes of phase were less than 0.1 degree in both gas phase and liquid phase respectively. The same concentration of probe and target sequence hybridized in the network analyzer and the NI signal acquisition system, respectively, phase changes were 1.3 degree. NI signal acquisition system can achieve the requirements of the experiment.2. The optimal liquid phase hybridization conditions of two-terminal resonant-type LSAW sensor were included pH value of 7.6, ion concentration of 0.3mol/L, immobilized DNA probe concentration of 0.5μmol/L, target sequence concentration of 1.0μmol/L. Equilibrium time of hybridization reaction was 33min.3. By enzyme biological signal amplification, the biotin-labeled probe significantly increased the phase change, phase increased about 16 times, while biotin-unlabeled probe had very small phase changes.4. LSAW-SNP gene sensor results had shown target-G could result in significant phase changes, 21 times than that of the target-A. Detection of the amplified signal could be reduced to 20min. Of different concentrations of target-G test, the minimum detection limit was 1×10-12mol/L. The logarithm of target concentration in the range of 1×10-12mol/L 1×10-6mol/L and phase change had a good linear relationship, linear regression equation wasΔP =1.853IgC+22.235 with the correlation coefficient of 0.976.5. SA14-14-2 target sequence of phase changed significantly, while for P3 target is very small, coinciding with the gene sequencing results.Conclusions1. The introduction of reflexed bar array increased useful signal detection, improved two-terminal resonant-type LSAW sensor can satisfy needs of experimental detecting and research.2. Enzyme biological signal amplification system can significantly increase phase changes, effectively improve the signal to noise ratio and sensitivity when LSAW gene sensor is used to detect the target DNA sequence. It provides a reliable method of signal amplification for detecting biological response using biosensor implying great potential applications of biosensors.3. Based on DNA ligase reaction and enzyme signal amplification system, LSAW-SNP gene sensor detection system shows high specificity, sensitivity and accuracy through the actual sample detection of the Japanese encephalitis virus SNP site (A2293G). Therefore provides a fast, convenient, economical and practical method for clinical SNP detection. |
PDF Full Text Request