Construction Of Leaky Surface Acoustic Wave Bis-PNA Biosensor And The Detection Of HPV

Objectives:The aim of this study was to construct a leaky surface acoustic wave (LSAW) bis-PNA biosensor detection system to rapidly detect the target dsDNA, and to examine the superiority of the system by detecting the HPV.Methods:1. Technology of fine processing was firstly used to make the LSAW biosensor with double two-port resonatoras style. Then we developed the dual channels resonance circuit, the BSMS 1.0 software, and temperature controlling system. The detection system of LSAW biosensor was constructed by combining all these components with network analyzer and computer.2. HPV probe was immobilized on the gold electrode surface of the LSAW biosensor by thiol method and hybridized with complementary target sequence. The phase shift of hybridization reaction was observed. The sensitivity and the linear range were confirmed by analyzing the correlation between the phase shift and the concentration of the target sequence.3. According to the wavelet signal singularity detection and the phase shift during 10min, the reaction starting point and the reaction ending point of the LSAW biosensor were judged, and the criterion was established.4. The sensitivity, specificity, precision and reproducibility of LSAW biosensor were investigated. Then, the LSAW biosensor was used to directly detect the extracted genomic DNA from positive clinical samples diagnosed for HPV infection and negative control samples.5. To improve the sensitivity of the LSAW biosensor,"RecA protein-complementary single strand DNA probe"complex was used as biological signal amplification system. The optimization on the concentration of RecA protein and ATPγS was explored. Results:1. The signal reflex distance between import and export interdigital transducer was increased by the reflex bar of double two-port resonatoras of the LSAW biosensor. So the phase shift caused by the hybridized reaction was significantly improved, and the equilibrium time of hybridization was shortened.2. The dual channel circuit developed in our team was of strongly vibromotive ability in liquid phase and its stability of phase could also meet the need of our experiment. And the introduction of the temperature controlling system provided the optimal temperature environment for biological reaction.3. According to the wavelet transformation and the wavelet signal singularity detection, the phase shift was analysised at six frequency domain grades. The maximum of wavelet transformation was appeared when the signal mutated. Therefore, the reaction starting point of the LSAW biosensor was accurately judged.4. The phase shift (△P)≤0.2degin 10min was set to the detection system. At the end of the reaction, the system would blow a whistle automatically. So, the reaction ending point of the LSAW biosensor was accurately judged.5. The phase shift firstly enhanced and then tended gently for hybridization with HPV target DNA when concentration of target varied from 1pg/L to 1mg/L, and 100μg/L was the saturation point. The statistic linear regression equation was△P=0.3010 IgC+2.1753 among the range of target concentration from 1pg/L to 100μg/L and the correlating coefficient was 0.9932.6. The capability of recognizing mismatched base by bis-PNA probe was higher and the time spent on hybridization was shorter than that of DNA probe.7. After five regenerating cycles,the biosensor could retain 89.60% of the original response, and the coefficient of variance (CV) was 4.24%. After ten regenerating cycles the biosensor retained 71.56% of the original response and the CV was 10.14%. Thus, our biosensor was of good reproducibility.8. The intraassay and interassay CV for the LSAW biosensor were 5.99% and 7.14% for 10.00μg/L, 6.34% and 7.09% for 100.00μg/L, 6.76% and 8.44% for 1000.00μg/L respectively. The intraassay mean CV was 6.36% and the interassay mean CV was 7.56%. Both the CVs were lower than 10%. 9. Among 36 clinical cases being diagnosed for HPV infection by PCR, 35 samples were detected correctly by the LSAW biosensor. The positive rate was 97.22%. The DNA concentrations of HPV in the 36 clinical cases varied from 1.40 x 102 to 9.11 x 107copies per milliliter clinical sample (i.e., 1.21pg/L to 0.78μg/L). The HPV 18 was not detected in the 14 controls with either of two methods. The range of phase shift was from 1.1 deg to 4.5 deg and the time consumed was from 38 min to 75 min. Detection limit of the LSAW biosensor was 1.40 x 102 copies per milliliter clinical sample (i.e., 1.21 pg/L), which was calculated by quantitative PCR. No significant difference existed between the two methods according to the McNemar test. There existed significant consistency between the two methods according to the Kappa test.10. The phase shift was significantly obvious when the concentration of RecA protein was 45μg/mL and ATPγS was 2.5 mmol/L, compared with other concentrations (P<0.01). The value of phase shift was (11.74±1.03) deg. At the best concentration of RecA protein, the ratio of the phase shift and the time spent on hybridization obviously outclassed the other concentrations. Its phase shift, signal noise ratio and detectability were more superior to the direct hybridization of the bis-PNA probe and the target DNA sequence.Conclusions:1. The LSAW biosensor with dual resonance style was predominant for the detection of molecular biological experiments in liquid phase. The introduction of reflex bar array efficiently increased the signal noise ratio and hence increased the sensitivity of the sensor.2. The improved LSAW sensor detection system was suitable to the experimental requires in every parts including oscillation circuit, temperature controlling system and the software BSMS 1.0.3. According to the wavelet transformation and the wavelet signal singularity detection, and the base of MATLAB emulation, the maximum of phase shift was obtained, and the mutational site of phase shift was detected. Therefore, the reaction starting point of the LSAW biosensor was accurately determined.4. The software on judging ending points worked according to the difference (△P) of phase shift in 10min. The calculation equation of△P was△P=△Pn-△P(n-9). When△P≤0.2deg, the detection system would blow a whistle automatically. So, the reaction ending point of the LSAW biosensor was accurately judged. 5. The detectability of the LSAW biosensor was 1.2pg/L. It was greatly more sensitive than bulk acoustic wave biosensor. It could quickly detect pathogenic microorganism in microcontent.6. The capability of bis-PNA probe on distinguishing matched and mismatched base was higher than that of DNA/DNA, while the tolerant degree of bis-PNA probe on hybridizing with noncomplementary target DNA sequence was lower than that of DNA/DNA. Therefore, bis-PNA probe could improve the detection specificity of the LSAW biosensor.7. Piranha solution,1M HCL and 1M NaOH can be used to wash everything covered on the reaction area. The reaction area of the LSAW biosensor and IDTs is covered by gold on the surface of LiTaO3 crystal. Thus, the LSAW biosensor can not be injured by the cleaning process. The LSAW biosensor has good reproducibility, which can reduce the cost of detection.8. The intraassay CV showed very low SD(mean CV=6.36%) and the interassay CV had a slightly higher SD(mean CV=7.56%). However, they were both lower than 10%. Therefore, our study indicated that the stability of the LSAW biosensor system was satisfactory.9. HPV genomic DNA from clinical samples could be directly detected with bis-PNA probe. Sensitivity and specificity of the LSAW biosensor showed no significant difference when it compared with quantitative PCR, but less time was consumed and the probe no labeling at the LSAW biosensor.10. The sensitivity was effectively improved and the detection time was significantly shortened by applying"RecA protein-complementary single strand DNA probe"complex to the LSAW biosensor.