压电生物传感器检测单核苷酸多态性的实验研究和临床应用
摘要
一、研究背景和目的
近年来,压电生物传感器检测技术逐渐成为生物分析中的研究热点。它具有实时检测、准确定量、特异性高、无需标记、经济性等优点,被认为是现代临床诊断发展的一个新方向。其原理在于压电晶体表面负载的质量变化可通过自身谐振频率变化反映出来,并通过固定不同识别元件,能够检测DNA、抗原-抗体、酶、细菌等多种物质,在分子识别、临床诊断、分子成膜过程、表面性质、毒理研究等领域中已得到较为广泛的应用。同时压电生物传感器操作简便,环境要求简单,检测设备易于微型化使其具有更大的临床应用价值。
SNP (single nucleotide polymorphism,单核苷酸多态性)是指基因组特定位置上由单个核苷酸的变异所引起的一种DNA序列多态性,如果人群中这种变异的频率大于1%,即称为SNP。作为基因变异的主要形式,SNPs在对疾病的起因、风险评估和防治方面有着巨大的潜力。其研究已成为人类后基因组时代的主要内容之一。近年大量研究证实,SNP位点不仅与一些人类疾病直接相关、还可以用来预测个体对药物治疗的应答以及作为易患某种疾病的危险人群的标记,对疾病的风险评估和个性化医疗十分重要。因此,SNP的检测技术对于临床诊断医学具有重要的潜在意义,有望为临床疾病风险评估以及个性化医疗诊断的研究开辟新的切入点和研究焦点。
关于SNP的大量研究已经鉴定发现了很多和疾病相关的位点,但由于缺乏简单有效快速低廉的临床检测手段,SNP的研究还停留在实验室阶段,SNP潜在应用价值远远没有得到有效地挖掘。而压电生物传感器具有简单实用快速低廉的特点,如能将其应用于SNP的检测将具有广阔的潜在临床应用前景。
但SNP只有一个碱基的差异,SNP的检测需要检测方法能从样本中分辨出只有一个碱基差异的目的序列。靶序列和干扰序列之间仅有一个碱基的差异,质量变化小。这给基于质量效应原理的压电生物检测体系带来挑战,需要传感器具有更高的敏感性和更好的特异性。因为本研究拟首先从多个方面提高压电生物传感器的稳定性,然后通过试验证实生物素-链霉亲和素放大系统可以放大压电生物传感器的信号,提高其检测敏感性。在此基础上,建立高敏感的压电SNP检测方法。同时利用EIS方法对压电SNP检测体系进行评估和验证,并对实验条件进行优化。最后,以慢性乙肝易感相关SNP位点为靶点,初步研究压电SNP检测方法在临床标本检测中的应用情况。
二、方法和结果
1.利用酶生物信号放大系统建立高敏感的压电传感器SNP检测方法:
a.探讨生物素-链霉亲和素放大系统用来放大压电生物传感器的检测信号的可行性研究:分别固定标记生物素和未标记生物素的探针于压电传感器基片上,然后加入结合HRP的链亲和素以及DAB底物,检测分析频率变化。结果发现生物素将HRP-SA结合到基片上后,后者催化DAB形成的不可溶沉淀紧密附着于压电基片表面,引发谐振频率显著改变,最低可检测ssDNA片段10-13mol/l,而对照则无法检测出有意义的频率变化。表明酶生物信号放大系统可以用于压电传感器,对发生在后者之上的生物反应信号进行放大,可以有效提高压电传感器的检测敏感性。
b.利用酶生物信号放大系统,结合单碱基延伸技术,建立压电生物传感器的SNP检测体系:分别合成两条寡核苷酸,两者间仅存在一个碱基的不同(A/G);将其加入固定有未标记探针的压电检测池,分别利用Taq、Pfu以及Klenow fragment三种不同的DNA聚合酶将biotin-dUTP延伸结合到探针上;最后加入DAB,检测频率变化。结果表明含A碱基的序列能引起频率的显著变化,约20倍高于含G碱基的序列;单碱基延伸所采用的三种聚合酶中,pfu的特异性和敏感性最好。在模式试验中,本方法最低可检测出的最低阳性靶序列的浓度为2×10-11mol/l。与检测普通杂交信号比较,采用放大系统后,信号检测时间可缩短至20min,且结果稳定性更好。
2. EIS系统的构建及其应用:
将压电传感单元、三电极系统和电化学工作站共同组成EIS检测系统。设定适合分析压电基片表面金膜的电化学参数,对空白时、探针固定后及杂交后金膜表面进行了交流阻抗谱的检测,根据阻抗值分析金膜表面生物学反应情况。实验发现少部分实验用的压电基片空白表面交流阻抗值异常,可影响实验结果的重复性。探针固定和靶序列杂交的实验条件为:针对实验用的压电基片金膜面积而言,最适探针固定浓度为2μmol/l,最适固定时间为60min,靶序列最适杂交时间为60min。在压电传感器检测SNP的每一步骤中,金膜表面交流阻抗值均有相应变化。
3.压电SNP检测方法的临床应用:
首先以慢性乙肝持续感染相关SNP位点(ESR1 T392C)为靶点,分析并建立适合该位点的SNP检测探针、引物以及标准检测体系。然后收集的临床标本,提取基因组DNA,并进行特异性的扩增。采用压电SNP检测方法对其进行检测。结果表明该方法可以成功检测出临床来源标本中的ESR1 T392C位点。随后以经典的RFLP方法为参照方法,以RFLP检测的27例纯合子为样本,对比分析发现压电SNP检测方法TT型检出率为94.7%,CC型检出率为87.5%。两种方法经x2检验,p>0.05,表明我们建立的SNP压电检测方法在临床标本的实际检测中与RFLP相比具有较好的一致性。
三、结论:
1.酶生物放大系统可以显著增加压电传感器检测DNA靶序列时的频率变化,和干扰对照序列相比具有良好的特异性。表明酶生物放大系统有望作为一种特异、有效的放大手段应用于压电生物传感器,在检测微小质量变化样本中发挥重要作用。
2. EIS是一种有效的方法,可用来评价压电生物传感器,也可以用来优化其检测条件,其建立的实验条件符合压电检测实际情况,可用于压电生物传感器常规检测的标准步骤和标准体系的建立。
3.建立了压电SNP传感器检测慢性乙肝持续感染相关SNP位点(ESR1 T392C)的反应体系,初步研究了其频率变化特点;通过临床标本检测,并和传统方法RFLP对比分析,表明我们建立的SNP压电检测方法适合用于临床标本的实际检测。
Backgrounds
Recent 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 Results
1. Use enzyme signal amplification system to establish a high sensitivity Piezoelectric SNP biosensor
a) 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.
Conclusions
1. 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.
近年来,压电生物传感器检测技术逐渐成为生物分析中的研究热点。它具有实时检测、准确定量、特异性高、无需标记、经济性等优点,被认为是现代临床诊断发展的一个新方向。其原理在于压电晶体表面负载的质量变化可通过自身谐振频率变化反映出来,并通过固定不同识别元件,能够检测DNA、抗原-抗体、酶、细菌等多种物质,在分子识别、临床诊断、分子成膜过程、表面性质、毒理研究等领域中已得到较为广泛的应用。同时压电生物传感器操作简便,环境要求简单,检测设备易于微型化使其具有更大的临床应用价值。
SNP (single nucleotide polymorphism,单核苷酸多态性)是指基因组特定位置上由单个核苷酸的变异所引起的一种DNA序列多态性,如果人群中这种变异的频率大于1%,即称为SNP。作为基因变异的主要形式,SNPs在对疾病的起因、风险评估和防治方面有着巨大的潜力。其研究已成为人类后基因组时代的主要内容之一。近年大量研究证实,SNP位点不仅与一些人类疾病直接相关、还可以用来预测个体对药物治疗的应答以及作为易患某种疾病的危险人群的标记,对疾病的风险评估和个性化医疗十分重要。因此,SNP的检测技术对于临床诊断医学具有重要的潜在意义,有望为临床疾病风险评估以及个性化医疗诊断的研究开辟新的切入点和研究焦点。
关于SNP的大量研究已经鉴定发现了很多和疾病相关的位点,但由于缺乏简单有效快速低廉的临床检测手段,SNP的研究还停留在实验室阶段,SNP潜在应用价值远远没有得到有效地挖掘。而压电生物传感器具有简单实用快速低廉的特点,如能将其应用于SNP的检测将具有广阔的潜在临床应用前景。
但SNP只有一个碱基的差异,SNP的检测需要检测方法能从样本中分辨出只有一个碱基差异的目的序列。靶序列和干扰序列之间仅有一个碱基的差异,质量变化小。这给基于质量效应原理的压电生物检测体系带来挑战,需要传感器具有更高的敏感性和更好的特异性。因为本研究拟首先从多个方面提高压电生物传感器的稳定性,然后通过试验证实生物素-链霉亲和素放大系统可以放大压电生物传感器的信号,提高其检测敏感性。在此基础上,建立高敏感的压电SNP检测方法。同时利用EIS方法对压电SNP检测体系进行评估和验证,并对实验条件进行优化。最后,以慢性乙肝易感相关SNP位点为靶点,初步研究压电SNP检测方法在临床标本检测中的应用情况。
二、方法和结果
1.利用酶生物信号放大系统建立高敏感的压电传感器SNP检测方法:
a.探讨生物素-链霉亲和素放大系统用来放大压电生物传感器的检测信号的可行性研究:分别固定标记生物素和未标记生物素的探针于压电传感器基片上,然后加入结合HRP的链亲和素以及DAB底物,检测分析频率变化。结果发现生物素将HRP-SA结合到基片上后,后者催化DAB形成的不可溶沉淀紧密附着于压电基片表面,引发谐振频率显著改变,最低可检测ssDNA片段10-13mol/l,而对照则无法检测出有意义的频率变化。表明酶生物信号放大系统可以用于压电传感器,对发生在后者之上的生物反应信号进行放大,可以有效提高压电传感器的检测敏感性。
b.利用酶生物信号放大系统,结合单碱基延伸技术,建立压电生物传感器的SNP检测体系:分别合成两条寡核苷酸,两者间仅存在一个碱基的不同(A/G);将其加入固定有未标记探针的压电检测池,分别利用Taq、Pfu以及Klenow fragment三种不同的DNA聚合酶将biotin-dUTP延伸结合到探针上;最后加入DAB,检测频率变化。结果表明含A碱基的序列能引起频率的显著变化,约20倍高于含G碱基的序列;单碱基延伸所采用的三种聚合酶中,pfu的特异性和敏感性最好。在模式试验中,本方法最低可检测出的最低阳性靶序列的浓度为2×10-11mol/l。与检测普通杂交信号比较,采用放大系统后,信号检测时间可缩短至20min,且结果稳定性更好。
2. EIS系统的构建及其应用:
将压电传感单元、三电极系统和电化学工作站共同组成EIS检测系统。设定适合分析压电基片表面金膜的电化学参数,对空白时、探针固定后及杂交后金膜表面进行了交流阻抗谱的检测,根据阻抗值分析金膜表面生物学反应情况。实验发现少部分实验用的压电基片空白表面交流阻抗值异常,可影响实验结果的重复性。探针固定和靶序列杂交的实验条件为:针对实验用的压电基片金膜面积而言,最适探针固定浓度为2μmol/l,最适固定时间为60min,靶序列最适杂交时间为60min。在压电传感器检测SNP的每一步骤中,金膜表面交流阻抗值均有相应变化。
3.压电SNP检测方法的临床应用:
首先以慢性乙肝持续感染相关SNP位点(ESR1 T392C)为靶点,分析并建立适合该位点的SNP检测探针、引物以及标准检测体系。然后收集的临床标本,提取基因组DNA,并进行特异性的扩增。采用压电SNP检测方法对其进行检测。结果表明该方法可以成功检测出临床来源标本中的ESR1 T392C位点。随后以经典的RFLP方法为参照方法,以RFLP检测的27例纯合子为样本,对比分析发现压电SNP检测方法TT型检出率为94.7%,CC型检出率为87.5%。两种方法经x2检验,p>0.05,表明我们建立的SNP压电检测方法在临床标本的实际检测中与RFLP相比具有较好的一致性。
三、结论:
1.酶生物放大系统可以显著增加压电传感器检测DNA靶序列时的频率变化,和干扰对照序列相比具有良好的特异性。表明酶生物放大系统有望作为一种特异、有效的放大手段应用于压电生物传感器,在检测微小质量变化样本中发挥重要作用。
2. EIS是一种有效的方法,可用来评价压电生物传感器,也可以用来优化其检测条件,其建立的实验条件符合压电检测实际情况,可用于压电生物传感器常规检测的标准步骤和标准体系的建立。
3.建立了压电SNP传感器检测慢性乙肝持续感染相关SNP位点(ESR1 T392C)的反应体系,初步研究了其频率变化特点;通过临床标本检测,并和传统方法RFLP对比分析,表明我们建立的SNP压电检测方法适合用于临床标本的实际检测。
Backgrounds
Recent 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 Results
1. Use enzyme signal amplification system to establish a high sensitivity Piezoelectric SNP biosensor
a) 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.
Conclusions
1. 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.
引文
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