基于磁球技术的DNA单分子的体积放大以及电化学检测
摘要
本论文分为二章,第一章是基于体积放大技术的DNA单分子目视检测;第二章是基于纳米磁球介导酶联DNA单分子电化学检测。
在第一章中我们提出了一种简单的基于体积放大技术的DNA单分子检测方法。该方法分为三步:首先利用生物素与链霉亲和素的特异性反应将生物素化的DNA捕捉探针固定在链霉亲和素修饰的聚乙烯板上,然后滴加目标DNA溶液,使其与捕捉探针杂交。第二步,取一定量链霉亲和素修饰的5.91μm的磁球与过量的生物素化的检测探针反应,利用磁分离除去过量的检测探针。最后,将第一、第二两步反应的产物按一定比例混合,进行第二次杂交反应,通过目标DNA与检测探针的杂交,使形成捕捉探针—目标—检测探针—磁球的结构,进而将磁球固定在聚乙烯板上。用倒置显微镜以×25倍或×40倍物镜用CCD照像或×250或×400倍用眼观察。如果在上面的第一步中,使固定在微孔板上的目标DNA足够稀,通过杂交反应最终一个磁球上只结合一个目标DNA分子,这样,通过对磁球的计数检测,就等于实现对单个DNA分子的计数检测。该方法的最大优点是不需用复杂而昂贵的仪器,只用普通的显微镜配合常用的CCD(也可用目视)就可进行单个目标DNA分子的检测。本章就杂交缓冲溶液的浓度、杂交方式、清洗次数和反应时间进行了讨论,得到了最佳的杂交实验条件。使用MetaMorph软件对磁球进行计数,实现了对目标DNA单分子计数检测,线性范围5×10~(-16)-1×10~(-14)mol/L。
在第二章中,建立了一种电化学检测单个DNA分子的新方法。该方法基于核酸功能化的纳米磁球实现DNA的初步放大,然后利用磁球表面的碱性磷酸酶的催化反应实现第二次信号放大,最后利用毛细管作为单分子取样通道并于柱端进行电化学检测。该方法具有以下几个优点:(1)灵敏度高。在本实验中,不是通过检测目标DNA而是碱性磷酸酶催化底物得到的产物苯酚,苯酚通过磁球技术的DNA放大以及酶放大两步放大反应得到,使检测信号大大增强。(2)重现性好。在电化学检测中电极表面的电化学活性的不稳定常会导致电信号的重现性变差,而本方法不是根据信号的强度,而是根据峰的个数来检测目标DNA,所以信号强度的重现性对实验不是很重要。这一特点使方法的重现性优于常规的电化学检测。(3)该实验不需要昂贵的仪器设备,磁球与链霉亲和素修饰的聚苯乙烯板都已经商品化。通过磁球技术与酶催化两步放大相结合实现了用电化学手段对单个DNA分子的定性及定量检测,检测线性范围5×10~(-16)-1×10~(-13)mol/L。
In chapter one of this thesis,we provided a novel method to detect single DNA molecules.A sandwich-type hybridization assay is performed on a streptavidin-coated substrate.A large number of biotinylated capture DNAs(c-DNAs)are first covalently bound to the substrate through the interaction between streptavidin and biotin.Then, the complementary target DNAs(t-DNAs)are hybridized with the c-DNAs attached to the substrate,followed by hybridization between t-DNAs and biotinylated first probe DNAs(p-DNAs)conjugated to streptavidin-coated magnetic microparticles (MMPs).In this case,one t-DNA binds one MMP which could be observed under an optical microscope.To quantitatively determine t-DNAs,The images of all MMPs were taken by DP70,then the number of MMPs on the images were obtained by using a MateMorph software.The number of the MMPs is linearly proportional to the concentration of t-DNA in a range of 5×10~(-16)-1×10~(-14)mol/L。Additionally,we disscuss the influence of hybridization time,hybridization form,hybridization buffered solution and the times of the cleaning to hybridization effect.This size amplification method was simple and could be applied to other biomolecules.
In chapter two,we describe a magnetic nanobead-based single-molecule electrochemical detection method of DNA by combining DNA amplification and enzyme-catalysis amplification.A sandwich-type hybridization assay is performed on a streptavidin-coated substrate.A large number of biotinylated capture DNAs (c-DNAs)are first covalently bound to the substrate through the interaction between streptavidin and biotin.Then,the complementary target DNAs(t-DNAs)are hybridized with the c-DNAs attached to the substrate,followed by hybridization between t-DNAs and biotinylated first probe DNAs(p-DNA1s)conjugated to streptavidin-coated magnetic nanobeads(MNBs).In this case,one t-DNA binds one MNB.Subsequently,the MNBs are released from the substrate and transferred to another vessel.Then,biotinylated second probe DNAs(p-DNA2s)are hybridized to the p-DNA1s on the surface of the MNBs.After magnetic separation from the reaction media,the biotinylated p-DNA2 on the MNBs are labeled with alkaline phosphatase(AP)conjugated by streptavidin through the interaction between streptavidin and biotin.The MNBs with DNA hybrid labeled by AP(AP-DNA-MNBs) with enzyme substrate disodium phenyl phosphate(DPP)are continuously introduced by pressure through a capillary as the microsampler and microreactor by means of a microsyringe pump.AP on the AP-DNA-MNBs converts a huge number of DPP into its product phenol around each AP-DNA-MNB during AP-DNA-MNB movement in the capillary.The phenol zones with the moving AP-DNA-MNBs are continuously delivered to the capillary outlet and detected by electrochemical detection.One phenol zone corresponding to one t-DNA produces one peak on elution curve.The number of the peaks is linearly proportional to the concentration of t-DNA in a range of 5.0×10~(-16)-1.0×10~(-13).The high sensitivity is because that in this method,phenol is detected rather than t-DNA.The phenol is generated through double amplification, one t-DNA molecule produces 7000 AP molecules on the surface of one AP-DNA-MNB(DNA amplification)and one AP molecule produces 5×10~4 phenol molecules(enzyme amplification).Therefore,an amplification factor of 10~8 is obtained in the method,which leads to that electrochemical technique can detect single DNA molecules.The quantitative method relies on counting the number of the peaks corresponding to single t-DNA molecules rather than signal intensity of the peaks.Thus,the detected signal intensity is not important.This feature guarantees the reliability of the electrochemically quantitative assay for DNA.Additionally,the selectivity of the method is high.
在第一章中我们提出了一种简单的基于体积放大技术的DNA单分子检测方法。该方法分为三步:首先利用生物素与链霉亲和素的特异性反应将生物素化的DNA捕捉探针固定在链霉亲和素修饰的聚乙烯板上,然后滴加目标DNA溶液,使其与捕捉探针杂交。第二步,取一定量链霉亲和素修饰的5.91μm的磁球与过量的生物素化的检测探针反应,利用磁分离除去过量的检测探针。最后,将第一、第二两步反应的产物按一定比例混合,进行第二次杂交反应,通过目标DNA与检测探针的杂交,使形成捕捉探针—目标—检测探针—磁球的结构,进而将磁球固定在聚乙烯板上。用倒置显微镜以×25倍或×40倍物镜用CCD照像或×250或×400倍用眼观察。如果在上面的第一步中,使固定在微孔板上的目标DNA足够稀,通过杂交反应最终一个磁球上只结合一个目标DNA分子,这样,通过对磁球的计数检测,就等于实现对单个DNA分子的计数检测。该方法的最大优点是不需用复杂而昂贵的仪器,只用普通的显微镜配合常用的CCD(也可用目视)就可进行单个目标DNA分子的检测。本章就杂交缓冲溶液的浓度、杂交方式、清洗次数和反应时间进行了讨论,得到了最佳的杂交实验条件。使用MetaMorph软件对磁球进行计数,实现了对目标DNA单分子计数检测,线性范围5×10~(-16)-1×10~(-14)mol/L。
在第二章中,建立了一种电化学检测单个DNA分子的新方法。该方法基于核酸功能化的纳米磁球实现DNA的初步放大,然后利用磁球表面的碱性磷酸酶的催化反应实现第二次信号放大,最后利用毛细管作为单分子取样通道并于柱端进行电化学检测。该方法具有以下几个优点:(1)灵敏度高。在本实验中,不是通过检测目标DNA而是碱性磷酸酶催化底物得到的产物苯酚,苯酚通过磁球技术的DNA放大以及酶放大两步放大反应得到,使检测信号大大增强。(2)重现性好。在电化学检测中电极表面的电化学活性的不稳定常会导致电信号的重现性变差,而本方法不是根据信号的强度,而是根据峰的个数来检测目标DNA,所以信号强度的重现性对实验不是很重要。这一特点使方法的重现性优于常规的电化学检测。(3)该实验不需要昂贵的仪器设备,磁球与链霉亲和素修饰的聚苯乙烯板都已经商品化。通过磁球技术与酶催化两步放大相结合实现了用电化学手段对单个DNA分子的定性及定量检测,检测线性范围5×10~(-16)-1×10~(-13)mol/L。
In chapter one of this thesis,we provided a novel method to detect single DNA molecules.A sandwich-type hybridization assay is performed on a streptavidin-coated substrate.A large number of biotinylated capture DNAs(c-DNAs)are first covalently bound to the substrate through the interaction between streptavidin and biotin.Then, the complementary target DNAs(t-DNAs)are hybridized with the c-DNAs attached to the substrate,followed by hybridization between t-DNAs and biotinylated first probe DNAs(p-DNAs)conjugated to streptavidin-coated magnetic microparticles (MMPs).In this case,one t-DNA binds one MMP which could be observed under an optical microscope.To quantitatively determine t-DNAs,The images of all MMPs were taken by DP70,then the number of MMPs on the images were obtained by using a MateMorph software.The number of the MMPs is linearly proportional to the concentration of t-DNA in a range of 5×10~(-16)-1×10~(-14)mol/L。Additionally,we disscuss the influence of hybridization time,hybridization form,hybridization buffered solution and the times of the cleaning to hybridization effect.This size amplification method was simple and could be applied to other biomolecules.
In chapter two,we describe a magnetic nanobead-based single-molecule electrochemical detection method of DNA by combining DNA amplification and enzyme-catalysis amplification.A sandwich-type hybridization assay is performed on a streptavidin-coated substrate.A large number of biotinylated capture DNAs (c-DNAs)are first covalently bound to the substrate through the interaction between streptavidin and biotin.Then,the complementary target DNAs(t-DNAs)are hybridized with the c-DNAs attached to the substrate,followed by hybridization between t-DNAs and biotinylated first probe DNAs(p-DNA1s)conjugated to streptavidin-coated magnetic nanobeads(MNBs).In this case,one t-DNA binds one MNB.Subsequently,the MNBs are released from the substrate and transferred to another vessel.Then,biotinylated second probe DNAs(p-DNA2s)are hybridized to the p-DNA1s on the surface of the MNBs.After magnetic separation from the reaction media,the biotinylated p-DNA2 on the MNBs are labeled with alkaline phosphatase(AP)conjugated by streptavidin through the interaction between streptavidin and biotin.The MNBs with DNA hybrid labeled by AP(AP-DNA-MNBs) with enzyme substrate disodium phenyl phosphate(DPP)are continuously introduced by pressure through a capillary as the microsampler and microreactor by means of a microsyringe pump.AP on the AP-DNA-MNBs converts a huge number of DPP into its product phenol around each AP-DNA-MNB during AP-DNA-MNB movement in the capillary.The phenol zones with the moving AP-DNA-MNBs are continuously delivered to the capillary outlet and detected by electrochemical detection.One phenol zone corresponding to one t-DNA produces one peak on elution curve.The number of the peaks is linearly proportional to the concentration of t-DNA in a range of 5.0×10~(-16)-1.0×10~(-13).The high sensitivity is because that in this method,phenol is detected rather than t-DNA.The phenol is generated through double amplification, one t-DNA molecule produces 7000 AP molecules on the surface of one AP-DNA-MNB(DNA amplification)and one AP molecule produces 5×10~4 phenol molecules(enzyme amplification).Therefore,an amplification factor of 10~8 is obtained in the method,which leads to that electrochemical technique can detect single DNA molecules.The quantitative method relies on counting the number of the peaks corresponding to single t-DNA molecules rather than signal intensity of the peaks.Thus,the detected signal intensity is not important.This feature guarantees the reliability of the electrochemically quantitative assay for DNA.Additionally,the selectivity of the method is high.
引文
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