漏声表面波—双体肽核酸生物传感器的构建及检测HPV的实验研究
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摘要
目的:
构建新型漏声表面波(LSAW)-双体肽核酸生物传感器检测系统,实现快速检测DNA双链病毒,并通过检测HPV验证传感器的优越性。
方法:
1.利用精细微加工方法制备双端对谐振型LSAW生物传感器,自行研发自激式振荡电路、新型温控系统以及BSMS 1.0数据采集分析软件,将它们与网络分析仪、GPIB控制卡及计算机联用以构建LSAW生物传感器检测系统。
2.用巯基末端修饰共价结合法将人乳头瘤病毒的特异性探针固定在LSAW生物传感器的金膜表面,并与互补的靶序列进行杂交反应。根据相位变化和靶序列浓度之间的对应关系确定传感器的检测灵敏度及检测范围。
3.根据小波奇性检测原理及连续十分钟内相位变化差值(△P)分别对传感器的反应起点和终点进行判断,并拟定判断标准。
4.通过系列实验,探讨传感器检测HPV基因组DNA的灵敏度、特异性、精密性及传感器的再生性等动态响应规律及性能参数,并对HPV感染引起的宫颈癌患者的生殖道分泌物进行了临床标本检测,同时与荧光定量PCR法进行方法学比较。
5.以“RecA蛋白-互补单链DNA”探针为生物信号放大系统提高传感器的检测灵敏度,在反应过程中加入ATPγS提供能量,分别探索优化RecA蛋白及ATPγS的最佳反应浓度。
结果:
1.双端对谐振型LSAW生物传感器的反射栅阵列增加了输入输出叉指换能器之间信号传播的行程,提高了杂交反应引起的相位变化值,缩短了杂交反应平衡时间。
2.自行研制的双通道振荡电路,在液相中起振非常容易,其相位稳定度达到实用要求。温度控制系统的引入提供了杂交反应的最佳温度环境。
3.利用小波变换和小波奇性检测原理对传感器采集到的相位信号进行6级频域分析,由于函数的奇异点可以从小波变换的模极大值检测出来,而小波变换的模极大值都是出现在信号有突变的地方,因此准确地判断了杂交反应的起始点。
4.将检测到的实验数据进行平均拟合,设定连续十分钟内相位变化差值(△P)≤0.2deg检测系统就会自动鸣笛报警,从而准确地确定了杂交反应的终点。
5.随着HPV靶序列浓度从1pg/L到1mg/L变化时,杂交反应所引起的相位变化值先上升后趋于饱和,以100μg/L为饱和点;在1pg/L到100μg/L的HPV靶序列浓度范围内,相位变化与靶序列浓度的线性回归方程为△P=0.3010 IgC+2.1753,相关系数R2=0.9932。
6.与普通DNA探针相比,bis-PNA探针对碱基错配的识别能力较强,即bis-PNA探针与靶序列DNA结合具有高度的特异性,且杂交时间更短。
7.传感器再生使用第5次时核酸杂交引起的相位变化值相当于第一次的89.60%,变异系数为4.24%;再生使用第10次时相位变化值相当于第一次的71.56%,变异系数为10.14%。
8. HPV DNA浓度分别为10.00、100.00、100.00μg/L时,天内和天间精密性实验的CV值分别为5.99%和7.14%,6.34%和7.09%,6.76%和8.44%。上述三种不同浓度HPV DNA的天内和天间精密性实验的平均CV值分别为6.36%和7.56%,均小于10%。
9. 36例荧光定量PCR检测HPV的阳性标本,其中35例经LSAW传感器检测为HPV 18阳性,阳性率为97.22%。临床标本中HPV DNA含量的变化范围是每毫升1.40×10~2拷贝至9.11×10~7拷贝(即1.21 pg/L至0.78μg/L),对应的相位变化范围是(1.1～4.5)deg,反应平衡时间为(38～75)min。在14例阴性对照标本中,两种方法的检测结果均为阴性。通过定量PCR检测结果换算得出传感器检测临床标本HPV的最低拷贝数为每毫升1.40×102拷贝,即1.21pg/L。经McNemanr统计分析传感器和PCR两种检测方法没有显著性差异。Kappa检验说明两种方法检测结果一致性很好。
10.当RecA蛋白浓度为45μg/ml,ATPγS浓度为2.5 mmol/L时,与其他浓度组相比,引起的相位变化值最大(P<0.01)为(11.74±1.03)deg。而且相位变化值与平衡时间的比值也远远高于其他浓度组,和bis-PNA探针与靶序列DNA直接进行杂交反应相比,传感器的响应相位、信噪比和检测灵敏度均有显著提高。
结论:
1.双端对谐振型LSAW生物传感器更适合液相中生物分子杂交反应的检测,反射栅阵列有效提高了传感器的信噪比和检测灵敏度。
2.自主构建的LSAW传感器检测系统在振荡电路、温控系统、数据采集分析等方面均达到试验要求。
3.利用小波变换和奇性检测原理,以MATLAB仿真为基础,获得了相位信号的小波变换模极大值图谱,检测出相位信号的突变点,从而准确地确定了反应起点。
4.自行编写的终点判断软件根据连续十分钟内相位变化差值(△P),即△P=△Pn-△P(n-9)≤0.2deg可以实时准确判断反应终点。
5. LSAW传感器检测限灵敏度可达到1pg/L级,与体波传感器相比检测灵敏度有了很大提高,能够实现更加微量病原微生物的快速检测。
6. bis-PNA探针能够精确区分完全互补及单碱基错配的核酸序列,对互补DNA的错配容忍程度比相应的DNA/DNA更低,bis-PNA作为杂交探针可极大提高LSAW生物传感器检测系统的特异性。
7.采用piranha液、1M HCL和1M NaOH等三种溶液连续洗脱,可以将LSAW传感器反应区域固定的所有物质洗脱掉。传感器的叉指换能器由硅橡胶密封,以保护叉指换能器电极,在清洗的过程对传感器的损伤很小。因而传感器具有较好的再生性能,可进一步降低临床检测应用中的成本。
8.三种不同HPV DNA浓度的天间精密性实验的平均CV值为7.56%,略高于天内精密性实验的平均CV值6.36%,但二者均小于10%。因此,LSAW传感器具有良好的稳定性,从而保证了检测结果的可靠性和重复性。
9. LSAW双体肽核酸生物传感器实现了对临床标本中HPV基因组DNA的直接检测。与定量PCR法相比,两种方法检测的灵敏度和特异性无显著性差异,但传感器法所用时间更短,且无需进行探针的标记。
10.“RecA蛋白-互补单链DNA”探针复合体与bis-PNA/dsDNA复合物相结合可以有效提高传感器的检测灵敏度,并明显缩短检测时间
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.
构建新型漏声表面波(LSAW)-双体肽核酸生物传感器检测系统,实现快速检测DNA双链病毒,并通过检测HPV验证传感器的优越性。
方法:
1.利用精细微加工方法制备双端对谐振型LSAW生物传感器,自行研发自激式振荡电路、新型温控系统以及BSMS 1.0数据采集分析软件,将它们与网络分析仪、GPIB控制卡及计算机联用以构建LSAW生物传感器检测系统。
2.用巯基末端修饰共价结合法将人乳头瘤病毒的特异性探针固定在LSAW生物传感器的金膜表面,并与互补的靶序列进行杂交反应。根据相位变化和靶序列浓度之间的对应关系确定传感器的检测灵敏度及检测范围。
3.根据小波奇性检测原理及连续十分钟内相位变化差值(△P)分别对传感器的反应起点和终点进行判断,并拟定判断标准。
4.通过系列实验,探讨传感器检测HPV基因组DNA的灵敏度、特异性、精密性及传感器的再生性等动态响应规律及性能参数,并对HPV感染引起的宫颈癌患者的生殖道分泌物进行了临床标本检测,同时与荧光定量PCR法进行方法学比较。
5.以“RecA蛋白-互补单链DNA”探针为生物信号放大系统提高传感器的检测灵敏度,在反应过程中加入ATPγS提供能量,分别探索优化RecA蛋白及ATPγS的最佳反应浓度。
结果:
1.双端对谐振型LSAW生物传感器的反射栅阵列增加了输入输出叉指换能器之间信号传播的行程,提高了杂交反应引起的相位变化值,缩短了杂交反应平衡时间。
2.自行研制的双通道振荡电路,在液相中起振非常容易,其相位稳定度达到实用要求。温度控制系统的引入提供了杂交反应的最佳温度环境。
3.利用小波变换和小波奇性检测原理对传感器采集到的相位信号进行6级频域分析,由于函数的奇异点可以从小波变换的模极大值检测出来,而小波变换的模极大值都是出现在信号有突变的地方,因此准确地判断了杂交反应的起始点。
4.将检测到的实验数据进行平均拟合,设定连续十分钟内相位变化差值(△P)≤0.2deg检测系统就会自动鸣笛报警,从而准确地确定了杂交反应的终点。
5.随着HPV靶序列浓度从1pg/L到1mg/L变化时,杂交反应所引起的相位变化值先上升后趋于饱和,以100μg/L为饱和点;在1pg/L到100μg/L的HPV靶序列浓度范围内,相位变化与靶序列浓度的线性回归方程为△P=0.3010 IgC+2.1753,相关系数R2=0.9932。
6.与普通DNA探针相比,bis-PNA探针对碱基错配的识别能力较强,即bis-PNA探针与靶序列DNA结合具有高度的特异性,且杂交时间更短。
7.传感器再生使用第5次时核酸杂交引起的相位变化值相当于第一次的89.60%,变异系数为4.24%;再生使用第10次时相位变化值相当于第一次的71.56%,变异系数为10.14%。
8. HPV DNA浓度分别为10.00、100.00、100.00μg/L时,天内和天间精密性实验的CV值分别为5.99%和7.14%,6.34%和7.09%,6.76%和8.44%。上述三种不同浓度HPV DNA的天内和天间精密性实验的平均CV值分别为6.36%和7.56%,均小于10%。
9. 36例荧光定量PCR检测HPV的阳性标本,其中35例经LSAW传感器检测为HPV 18阳性,阳性率为97.22%。临床标本中HPV DNA含量的变化范围是每毫升1.40×10~2拷贝至9.11×10~7拷贝(即1.21 pg/L至0.78μg/L),对应的相位变化范围是(1.1～4.5)deg,反应平衡时间为(38～75)min。在14例阴性对照标本中,两种方法的检测结果均为阴性。通过定量PCR检测结果换算得出传感器检测临床标本HPV的最低拷贝数为每毫升1.40×102拷贝,即1.21pg/L。经McNemanr统计分析传感器和PCR两种检测方法没有显著性差异。Kappa检验说明两种方法检测结果一致性很好。
10.当RecA蛋白浓度为45μg/ml,ATPγS浓度为2.5 mmol/L时,与其他浓度组相比,引起的相位变化值最大(P<0.01)为(11.74±1.03)deg。而且相位变化值与平衡时间的比值也远远高于其他浓度组,和bis-PNA探针与靶序列DNA直接进行杂交反应相比,传感器的响应相位、信噪比和检测灵敏度均有显著提高。
结论:
1.双端对谐振型LSAW生物传感器更适合液相中生物分子杂交反应的检测,反射栅阵列有效提高了传感器的信噪比和检测灵敏度。
2.自主构建的LSAW传感器检测系统在振荡电路、温控系统、数据采集分析等方面均达到试验要求。
3.利用小波变换和奇性检测原理,以MATLAB仿真为基础,获得了相位信号的小波变换模极大值图谱,检测出相位信号的突变点,从而准确地确定了反应起点。
4.自行编写的终点判断软件根据连续十分钟内相位变化差值(△P),即△P=△Pn-△P(n-9)≤0.2deg可以实时准确判断反应终点。
5. LSAW传感器检测限灵敏度可达到1pg/L级,与体波传感器相比检测灵敏度有了很大提高,能够实现更加微量病原微生物的快速检测。
6. bis-PNA探针能够精确区分完全互补及单碱基错配的核酸序列,对互补DNA的错配容忍程度比相应的DNA/DNA更低,bis-PNA作为杂交探针可极大提高LSAW生物传感器检测系统的特异性。
7.采用piranha液、1M HCL和1M NaOH等三种溶液连续洗脱,可以将LSAW传感器反应区域固定的所有物质洗脱掉。传感器的叉指换能器由硅橡胶密封,以保护叉指换能器电极,在清洗的过程对传感器的损伤很小。因而传感器具有较好的再生性能,可进一步降低临床检测应用中的成本。
8.三种不同HPV DNA浓度的天间精密性实验的平均CV值为7.56%,略高于天内精密性实验的平均CV值6.36%,但二者均小于10%。因此,LSAW传感器具有良好的稳定性,从而保证了检测结果的可靠性和重复性。
9. LSAW双体肽核酸生物传感器实现了对临床标本中HPV基因组DNA的直接检测。与定量PCR法相比,两种方法检测的灵敏度和特异性无显著性差异,但传感器法所用时间更短,且无需进行探针的标记。
10.“RecA蛋白-互补单链DNA”探针复合体与bis-PNA/dsDNA复合物相结合可以有效提高传感器的检测灵敏度,并明显缩短检测时间
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.
引文
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