基于邻近表面杂交分析的电化学生物传感技术用于蛋白和核酸的检测
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摘要
随着人类基因组计划的完成和功能蛋白质组学、基因组学的研究进展,发展高灵敏的方法用于蛋白质和核酸的快速分子监测就成为后基因时代的主要研究领域。电化学传感器由于其仪器简单、灵敏、选择性高等优点吸引了许多研究人员的注意,并成为研究的热点。
基于当前对蛋白质和核酸的检测技术提出的高灵敏、高选择、高特异性和简便、高通量的要求,本论文发展了一系列新的电化学生物传感方法用于检测蛋白质和核酸,为蛋白质和核酸分子检测提供高性能的技术平台。并通过对实际样品的分析以及与经典检测方法的结果对照,初步验证了这些技术的实用性。
(1)在第2章,我们利用核酸适体这一种通过体外组合化学筛选技术所得到的对目标蛋白分子具有高亲和力的寡核苷酸探针,基于一对亲合性核酸适体探针同时识别目标分子而空间邻近导致与某一互补序列杂交使稳定性提高这一邻近效应,提出了一种高灵敏的检测模型分析物血小板衍生生长因子BB(PDGF-BB)的表面邻近杂交分析电化学适体传感方法。该法首先在金电极表面利用巯基自组装技术固定了巯基修饰的短链寡核苷酸探针,设计了一个尾端延伸序列与短链寡核苷酸探针互补的PDGF-B核酸适体探针。该适体探针尾端修饰电活性基团二茂铁,且单个尾端序列与表面固定的短链寡核苷酸探针的熔链温度较低。利用该核酸适体探针可成对地与二聚体目标蛋白PDGF-BB同时结合,由于邻近效应促进两尾端序列同时与电极表面固定的两条短链寡核苷酸杂交,使得适体探针末端二茂铁标记与电极充分接近而发生高效电子传递,产生了显著的氧化还原电流。我们运用循环伏安法、差示脉冲伏安法、阻抗谱对该法进行了系统的表征,证实了该传感机理。结果表明,该法可实现PDGF-BB的高特异性地检测,响应动态范围可从1.0 pg/mL到20 ng/mL,检测限可达1.0 pg/mL。
(2)鉴于上述邻近表面杂交分析技术中涉及一对邻近探针同时与第三探针的杂交,我们在实验中发现这种邻近杂交对热稳定性的改善仍然不是充分大,不利于分析灵敏度的提高与探针的设计。第3章中,我们在此实验发现的基础上,基于一对不同的核酸适体探针与目标蛋白同时结合而彼此邻近的效应,进一步发展了相关模型蛋白PDGF-BB检测的电化学适体表面邻近杂交分析传感技术。该法首先在金电极表面利用巯基自组装技术固定了巯基修饰的短链寡核苷酸探针,设计了一个尾端延伸序列与短链寡核苷酸探针5’半片段互补的PDGF-B核酸适体探针,该适体探针尾端修饰电活性基团二茂铁,且单个尾端序列与表面固定的短链寡核苷酸探针的熔链温度较低。同样地,设计了另一个尾端延伸序列与短链寡核苷酸探针3’半片段互补且熔链温度较低的PDGF-B核酸适体探针,其识别位点与第一适体探针不同,并且尾端序列下游具有与第一核酸适体探针尾端上游有较短的互补序列,这一短互补序列熔链温度也较低。两条不同的核酸适体探针可同时识别PDGF不同位点由于邻近而相互杂交,杂交体两尾端序列可与电极表面固定的巯基短链寡核苷酸杂交,并使末端二茂铁标记与电极充分接近,产生了显著的氧化还原电流。结果表明,该方法检测范围更宽,从0.1 pg/mL到50 ng/mL,检测限降低了一个数量级,为0.1 pg/mL。
(3)第4章中,鉴于表面邻近杂交分析的高特异性与高灵敏度,我们发展了目标核酸分子检测的表面邻近杂交电化学传感技术,进一步拓展了表面邻近杂交分析的应用范围。与蛋白分子表面邻近杂交不同的是,核酸分子邻近杂交可利用待测核酸序列本身构成一对邻近探针。为此,我们设计了一个寡核苷酸检测探针,其3’末端修饰二茂铁,5’半片段与目标核酸分子互补且具有较高熔链温度,若目标核酸分子存在,检测探针与其杂交形成稳定的杂交体,杂交体一侧检测探针与目标核酸分子其余序列邻近,促进与电极表面固定的巯基短链寡核苷酸杂交,并使末端二茂铁标记与电极充分接近,产生了显著的氧化还原电流。与传统夹心式杂交分析相比,该法保证了末端二茂铁标记与电极充分接近,电子转移效率提高,可望显著改善灵敏度。且由于邻近分析使用了更短的互补序列,可有效提高其选择性。结果表明,该方法检测的线性范围宽,为1 fM到1 nM,而且能够成功的识别不同碱基数错配的序列。
(4)第5章中,鉴于目前核酸适体较为有限不能满足大量蛋白检测的需要,我们发展了目标蛋白分子检测的表面邻近杂交电化学免疫传感技术,用一对核酸探针修饰的抗体代替核酸适体探针,将表面邻近杂交分析拓展运用于蛋白质的免疫检测,为建立蛋白分子检测的通用技术平台提供技术依据。以前列腺特异性抗原PSA为模型分析物,提出了基于表面邻近杂交分析的高灵敏PSA的电化学免疫传感技术。此技术原理与第3章的电化学适体传感器类似,利用一对识别PSA不同抗原决定簇的单克隆抗体,设计了与这一对单克隆抗体共价交联的两个寡核苷酸探针,一个尾端延伸序列与巯基修饰短链寡核苷酸探针5’半片段互补且熔链温度较低,该探针3’端修饰二茂铁;另一个尾端延伸序列与巯基修饰短链寡核苷酸探针3’半片段互补且熔链温度较低,而且,第二探针尾端序列下游与与第一探针尾端上游有较短的互补序列,这一短互补序列熔链温度也较低。抗体对与抗原同时结合后,两核酸探针由于邻近而相互杂交,杂交体两尾端序列可与电极表面固定的巯基短链寡核苷酸杂交,并使末端二茂铁标记与电极充分接近,产生了显著的氧化还原电流。结果表明,该方法检测动态范围为25 pg到1 ng,检测下限为25 pg,且该免疫传感器可再生使用。
(5)第6章中,提出了一种基于分子信标变构控制的电化学通道法检测目标核酸分子杂交反应的电化学DNA传感方法。该方法设计运用了一种发夹结构核酸探针分子,其一端标记有生物素作为阻塞源,另一端标记氨基并通过二硫杂环化合物共价固定于金电极表面,电极用巯基十一酸自组装膜封闭。由于发夹探针茎部核酸双链螺旋体空间位阻较大,生物素阻塞元与巯基十一酸疏水烷基链作用进一步提高空间位阻,不利于电活性分子表面扩散。当探针与目标核酸分子杂交时,探针发夹构象改变,茎部双链结构改变为柔性单链结构,阻塞物离开电极表面,在巯基十一酸自组装膜内形成电化学针孔通道,使得电活性分子可进行表面扩散,产生电化学氧化还原电流。使用该方法对包含第142位密码子的α地中海贫血基因片断进行了分析。浓度检测动态线性范围为2.8×10-18 - 8.7×10-8 M,检测限可达1.1×10-19 M。该方法灵敏、特异性好、无需标记、可再生,可以很好地用于单碱基错配的区分。
(6)第7章中,建立了一种基于纳米金标记及金增强表面吸附伏安分析的新型电化学免疫传感技术。该方法以人免疫球蛋白G(h IgG)为模型分析物,将羊抗人免疫球蛋白G抗体固定于玻碳电极表面构成免疫传感器界面。固定化抗体与分析目标物h IgG发生免疫反应而将h IgG捕获,利用夹心法将纳米金标记的h IgG抗体结合于电极表面。通过纳米金自催化还原可在电极表面形成大粒径纳米金颗粒,增加电极表观面积与纳米金粒子与电极界面的电荷转移效率。利用电活性探针在金表面的自组装吸附,用差示脉冲伏安法对电极表面吸附量进行检测,可实现目标蛋白的高灵敏定量免疫分析。
With the implement of the Human Genome Project and the development of the functional genomics and proteomics research, it’s an important domain that high sensitive assay methods were developed for the fast detection of the proteins and DNA in real time in the after-genome age. Electochemical biosensors attract significant attention and become the research hotspot for their simple, sensitive, selective and compatible with microfabrication technologies, et al.
In this thesis, a series of novel electrochemical biosensor methods were developed to satisfy and achieve the need of the high sensitivity, selectivity and affinity, as well as prove a high performance platform for the detection of proteins and DNA. The results primarily proved that the proposed technology is reasonably comparable with the classical detection method, indicating the practicability of using the proposed method in clinical diagnosis. The detailed content described as follows:
(1)In chapter 2, we exploited aptamer that was short DNA or RNA oligonucleotides selected by SELEX with high affinity for the target proteins to developed a surface proximity-dependent hybridization electrochemical aptasensor method for the high sensitive detection of model analyte PDGF-BB based on the proximity effect, that is to say a pair of affinity aptamer probes simutineously recognize the target molecules and form the proximity probes so that some complementary sequence hybridization enhanced the stability. the aptasensor was constructed by self-assembly of a short thiolated DNA oligonucleotide 1 on the gold electrode via the alkanethiol moiety at the 5’-terminal. A DNA aptamer 2 to PDGF-BB with a sequence extension at the 3’-end is used as the affinity probe. The aptamer probe has a electroactive ferrocene-labeled tail sequence that is complementary to the surface-tethered DNA strands with a predesigned low melting temperature. When aptamer pairs simultaneously bind to the homodimer of PDGF-B, the tail sequence are brought into close proximity with their local concentration increased substantially to allow the pair of tail sequences to hybridize together with the surface-tethered DNA strands. Then the ferrocene labels of the tail sequence are drawn close to the electrode surface and produce a detectable redox current. CVs, DPVs and impedances were used to systemly characteristic this method, conforming the mechanism of the biosensor. In conclusion, this method can be implemented to the high affinity detection of PDGF-BB, dynamically increased DPV current with increasing PDGF-BB concentration ranging from 1.0 pg/mL to 20 ng/mL, with a readly achieved detection limit of 1.0 pg/mL.
(2)Whereas the aforementioned technology refered pair of proximity probes simultineouly to hybridizing with the surface-tethered DNA, it was found from the experiment that the proximity-dependent surface hybridization could not sufficiently improved the thermal stability and favorably enhanced the sensitivity. In Chapter 3, based on the mentioned mechanism of proximity-dependent surface hybridization assay, an electrochemical aptasensor based on the proximity-dependent surface hybridization assay by pair of different aptamer probes simultaneously bind to the protein for the detection of the related model protein-PDGF-BB was ulteriorly developed. The aptasensor interface was fabricated by self-assembly of a short thiolated DNA oligonucleotide 1 on the gold electrode via the alkanethiol moiety at the 5’-terminal. DNA aptamer 3 and 4 to PDGF-BB with a sequence extension at the 3’-end and the 5’-end is used as a pair of affinity probes. One aptamer probe has a electroactive ferrocene-labeled tail sequence at the 5’-end that is complementary to the surface-tethered DNA strands with a predesigned low melting temperature. Samely, the other aptamer probe with different recognization location has a tail sequence at the 3’-end that is complementary to the surface-tethered DNA strands with a predesigned low melting temperature. These two different aptamer probes could simultaneously bind to the different location of PDGF-BB homodimer. The tail sequence are brought into close proximity with their local concentration increased substantially to hybridized each other and allow the pair of tail sequences to hybridize together with the surface-tethered DNA strands. Then the ferrocene labels of the tail sequence are drawn close to the electrode surface and produce a detectable redox current. As a result, this method was proved to be wider detection range from 0.1 pg to 50 ng and lower detection limit of 0.1 pg.
(3)In chapter 4, proximity-dependent surface hybridization assay was developed to detect DNA by electrochemical biosensor method based on the high affinity and sensitivity of proximity-dependent surface hybridization assay in order to extending the application range. Different to the proteins, the nucleic acid detection based on proximity-dependent surface hybridization exploited the target DNA per se to construct a pair of proximity probes. So an oligonucleotide as the detection probe has a electroactive ferrocene-labeled tail at the 3’-end that is complementary to the target nucleic acid with a predesigned low melting temperature by 5’-end fragment. In the presense of target DNA, the detection probe and the target nucleic acid formed the hybridization complex. The hybridization one side was the detection probe that near to the other sequences of the target and make it hybridize to the surface-tethed oligonucleotide. This draw the ferrocene closed to the electrode surface and regenerated a readily detectable signal. Compare with the conventional sandwich hybridization assay, this approach ensured the ferrocene marker to enough close the electrode surface and increased the charge transport efficiency as well as improved the sensitivity remarkably. The detectable range dynamictally was from 1 fM to 1 nM and in the range the peak current exhibits a linear correlation to the log of target DNA concentration, the limit of the detection is 1 pM. This electrochemical DNA sensor can be regenerated and discriminate the mismatch with different bases sequences.
(4)In chapter 5, because of the aptamer unsatisfied the need of plentiful proteins detection, electrochemical immunosensor based-on proximity-dependent surface hybridization assay was used to detect the proteins. Herein a pair of antibody- modified oligonucleotids replaced the aptamer probes extend proximity-dependent surface hybridization assay to the proteins immunoassay that might creat a universal methodology for developing high-performance biosensors in sensitive detection of proteins. Electrochemical immunosensor based on proximity-dependent surface hybridization assay for high sensitive PSA detection was proposed. Similarly to the electrochemical aptasensor mechanism in chapter 3, a pair of mono-antibodies recognized different antigenic determinants of PSA were used to covalently crosslink two oligonucleotides. One probe has a electroactive ferrocene-labeled tail sequence at the 3’-end that is complementary to the surface-tethered DNA strands at 5’-end with a predesigned low melting temperature. Samely, the other probe has a tail sequence at the 3’-end that is complementary to the surface-tethered DNA strands at 3’-end with a predesigned low melting temperature. Furthermore, the first probe at the backward tail sequence and the seconde probe at the foreward tail sequence have a short complementary region with a low melting temperature. Antibodies simultaneously bind to the antigene, two oligonucleotides tail sequences are brought into close proximity with their local concentration increased substantially to hybridized each other and allow the pair of tail sequences to hybridize together with the surface-tethered DNA strands. Then the ferrocene labels of the tail sequence are drawn close to the electrode surface and produce a detectable redox current. The PSA was determined in the range of 25 pg to 1 ng with the detection limit of 25 pg and the immunosensor could be reusable.
(5) In chapter 6, a new sensitive, selective, reagentless and reusable electrochemical DNA sensor for detection of hybridization based on electrochemical channels switched by allosteric molecular beacon was described. This electrochemical DNA sensor employs hairpin oligonucleotide labeled with biotin as a block at one tail and labeled with NH2 at the other tail that interacted with disulfide heterocyclic compound to fix on the gold electrode surface. Because molecular beacon-like DNA stem duplex has the biggest space hindrance and the biotin as a block interacted with the 11-mercaptoundecanoic acid (MUA) increase the space hindrance, the electroactive molecules could not commendably diffuse on the electrode surface. When the probes hybridized with the target nucleic acid, the block was moved away from the electrode surface and thus formed an electrochemical pinhole channel in the 11-mercaptoundecanoic acid (MUA) self-assemble film. So the electroactive molecule can diffused, regenerated the electrochemical redox signal. The DNA target, associating with theα-thalassemia gene containing the codon 142, was determined in the range of 2.8×10-18~8.7×10-8 M, and the detection limit can be reached 1.1×10-19 M. This electrochemical sensor can be realized the high sensitive, selective, reagentless and reusable DNA biosensor and uses for indentication of SNPs.
(6) In chapter 7, a novel electrochemical immunosensor was established to detect h IgG based on gold nanoparticles tag and enhancement by surface absorbtion voltaggram assay. h IgG as the model analyte, the immunosensor interface was fabricated by immobilized GAH IgG on the glass carbon electrode surface. The immobilized antibody reacted with the analyte target-the h IgG and form sandwich configuration by using the colloidal gold-label goat anti-human immunoglobulin G (GAH IgG) as the detection probe. The size of gold nanoparticles and the apparent area of the electrode was enhanced by nanogold catalyting the reduction of HAuCl4 on the electrode surface. The self-asseble absorption of electroactive probes on the gold nanoparticles surface was exploited to measure the absorption amount on the electrode surface by DPV. The developed method is expected to hold great promise in immunosensing due to the ease of implementation, high sensitivity and specificity.
基于当前对蛋白质和核酸的检测技术提出的高灵敏、高选择、高特异性和简便、高通量的要求,本论文发展了一系列新的电化学生物传感方法用于检测蛋白质和核酸,为蛋白质和核酸分子检测提供高性能的技术平台。并通过对实际样品的分析以及与经典检测方法的结果对照,初步验证了这些技术的实用性。
(1)在第2章,我们利用核酸适体这一种通过体外组合化学筛选技术所得到的对目标蛋白分子具有高亲和力的寡核苷酸探针,基于一对亲合性核酸适体探针同时识别目标分子而空间邻近导致与某一互补序列杂交使稳定性提高这一邻近效应,提出了一种高灵敏的检测模型分析物血小板衍生生长因子BB(PDGF-BB)的表面邻近杂交分析电化学适体传感方法。该法首先在金电极表面利用巯基自组装技术固定了巯基修饰的短链寡核苷酸探针,设计了一个尾端延伸序列与短链寡核苷酸探针互补的PDGF-B核酸适体探针。该适体探针尾端修饰电活性基团二茂铁,且单个尾端序列与表面固定的短链寡核苷酸探针的熔链温度较低。利用该核酸适体探针可成对地与二聚体目标蛋白PDGF-BB同时结合,由于邻近效应促进两尾端序列同时与电极表面固定的两条短链寡核苷酸杂交,使得适体探针末端二茂铁标记与电极充分接近而发生高效电子传递,产生了显著的氧化还原电流。我们运用循环伏安法、差示脉冲伏安法、阻抗谱对该法进行了系统的表征,证实了该传感机理。结果表明,该法可实现PDGF-BB的高特异性地检测,响应动态范围可从1.0 pg/mL到20 ng/mL,检测限可达1.0 pg/mL。
(2)鉴于上述邻近表面杂交分析技术中涉及一对邻近探针同时与第三探针的杂交,我们在实验中发现这种邻近杂交对热稳定性的改善仍然不是充分大,不利于分析灵敏度的提高与探针的设计。第3章中,我们在此实验发现的基础上,基于一对不同的核酸适体探针与目标蛋白同时结合而彼此邻近的效应,进一步发展了相关模型蛋白PDGF-BB检测的电化学适体表面邻近杂交分析传感技术。该法首先在金电极表面利用巯基自组装技术固定了巯基修饰的短链寡核苷酸探针,设计了一个尾端延伸序列与短链寡核苷酸探针5’半片段互补的PDGF-B核酸适体探针,该适体探针尾端修饰电活性基团二茂铁,且单个尾端序列与表面固定的短链寡核苷酸探针的熔链温度较低。同样地,设计了另一个尾端延伸序列与短链寡核苷酸探针3’半片段互补且熔链温度较低的PDGF-B核酸适体探针,其识别位点与第一适体探针不同,并且尾端序列下游具有与第一核酸适体探针尾端上游有较短的互补序列,这一短互补序列熔链温度也较低。两条不同的核酸适体探针可同时识别PDGF不同位点由于邻近而相互杂交,杂交体两尾端序列可与电极表面固定的巯基短链寡核苷酸杂交,并使末端二茂铁标记与电极充分接近,产生了显著的氧化还原电流。结果表明,该方法检测范围更宽,从0.1 pg/mL到50 ng/mL,检测限降低了一个数量级,为0.1 pg/mL。
(3)第4章中,鉴于表面邻近杂交分析的高特异性与高灵敏度,我们发展了目标核酸分子检测的表面邻近杂交电化学传感技术,进一步拓展了表面邻近杂交分析的应用范围。与蛋白分子表面邻近杂交不同的是,核酸分子邻近杂交可利用待测核酸序列本身构成一对邻近探针。为此,我们设计了一个寡核苷酸检测探针,其3’末端修饰二茂铁,5’半片段与目标核酸分子互补且具有较高熔链温度,若目标核酸分子存在,检测探针与其杂交形成稳定的杂交体,杂交体一侧检测探针与目标核酸分子其余序列邻近,促进与电极表面固定的巯基短链寡核苷酸杂交,并使末端二茂铁标记与电极充分接近,产生了显著的氧化还原电流。与传统夹心式杂交分析相比,该法保证了末端二茂铁标记与电极充分接近,电子转移效率提高,可望显著改善灵敏度。且由于邻近分析使用了更短的互补序列,可有效提高其选择性。结果表明,该方法检测的线性范围宽,为1 fM到1 nM,而且能够成功的识别不同碱基数错配的序列。
(4)第5章中,鉴于目前核酸适体较为有限不能满足大量蛋白检测的需要,我们发展了目标蛋白分子检测的表面邻近杂交电化学免疫传感技术,用一对核酸探针修饰的抗体代替核酸适体探针,将表面邻近杂交分析拓展运用于蛋白质的免疫检测,为建立蛋白分子检测的通用技术平台提供技术依据。以前列腺特异性抗原PSA为模型分析物,提出了基于表面邻近杂交分析的高灵敏PSA的电化学免疫传感技术。此技术原理与第3章的电化学适体传感器类似,利用一对识别PSA不同抗原决定簇的单克隆抗体,设计了与这一对单克隆抗体共价交联的两个寡核苷酸探针,一个尾端延伸序列与巯基修饰短链寡核苷酸探针5’半片段互补且熔链温度较低,该探针3’端修饰二茂铁;另一个尾端延伸序列与巯基修饰短链寡核苷酸探针3’半片段互补且熔链温度较低,而且,第二探针尾端序列下游与与第一探针尾端上游有较短的互补序列,这一短互补序列熔链温度也较低。抗体对与抗原同时结合后,两核酸探针由于邻近而相互杂交,杂交体两尾端序列可与电极表面固定的巯基短链寡核苷酸杂交,并使末端二茂铁标记与电极充分接近,产生了显著的氧化还原电流。结果表明,该方法检测动态范围为25 pg到1 ng,检测下限为25 pg,且该免疫传感器可再生使用。
(5)第6章中,提出了一种基于分子信标变构控制的电化学通道法检测目标核酸分子杂交反应的电化学DNA传感方法。该方法设计运用了一种发夹结构核酸探针分子,其一端标记有生物素作为阻塞源,另一端标记氨基并通过二硫杂环化合物共价固定于金电极表面,电极用巯基十一酸自组装膜封闭。由于发夹探针茎部核酸双链螺旋体空间位阻较大,生物素阻塞元与巯基十一酸疏水烷基链作用进一步提高空间位阻,不利于电活性分子表面扩散。当探针与目标核酸分子杂交时,探针发夹构象改变,茎部双链结构改变为柔性单链结构,阻塞物离开电极表面,在巯基十一酸自组装膜内形成电化学针孔通道,使得电活性分子可进行表面扩散,产生电化学氧化还原电流。使用该方法对包含第142位密码子的α地中海贫血基因片断进行了分析。浓度检测动态线性范围为2.8×10-18 - 8.7×10-8 M,检测限可达1.1×10-19 M。该方法灵敏、特异性好、无需标记、可再生,可以很好地用于单碱基错配的区分。
(6)第7章中,建立了一种基于纳米金标记及金增强表面吸附伏安分析的新型电化学免疫传感技术。该方法以人免疫球蛋白G(h IgG)为模型分析物,将羊抗人免疫球蛋白G抗体固定于玻碳电极表面构成免疫传感器界面。固定化抗体与分析目标物h IgG发生免疫反应而将h IgG捕获,利用夹心法将纳米金标记的h IgG抗体结合于电极表面。通过纳米金自催化还原可在电极表面形成大粒径纳米金颗粒,增加电极表观面积与纳米金粒子与电极界面的电荷转移效率。利用电活性探针在金表面的自组装吸附,用差示脉冲伏安法对电极表面吸附量进行检测,可实现目标蛋白的高灵敏定量免疫分析。
With the implement of the Human Genome Project and the development of the functional genomics and proteomics research, it’s an important domain that high sensitive assay methods were developed for the fast detection of the proteins and DNA in real time in the after-genome age. Electochemical biosensors attract significant attention and become the research hotspot for their simple, sensitive, selective and compatible with microfabrication technologies, et al.
In this thesis, a series of novel electrochemical biosensor methods were developed to satisfy and achieve the need of the high sensitivity, selectivity and affinity, as well as prove a high performance platform for the detection of proteins and DNA. The results primarily proved that the proposed technology is reasonably comparable with the classical detection method, indicating the practicability of using the proposed method in clinical diagnosis. The detailed content described as follows:
(1)In chapter 2, we exploited aptamer that was short DNA or RNA oligonucleotides selected by SELEX with high affinity for the target proteins to developed a surface proximity-dependent hybridization electrochemical aptasensor method for the high sensitive detection of model analyte PDGF-BB based on the proximity effect, that is to say a pair of affinity aptamer probes simutineously recognize the target molecules and form the proximity probes so that some complementary sequence hybridization enhanced the stability. the aptasensor was constructed by self-assembly of a short thiolated DNA oligonucleotide 1 on the gold electrode via the alkanethiol moiety at the 5’-terminal. A DNA aptamer 2 to PDGF-BB with a sequence extension at the 3’-end is used as the affinity probe. The aptamer probe has a electroactive ferrocene-labeled tail sequence that is complementary to the surface-tethered DNA strands with a predesigned low melting temperature. When aptamer pairs simultaneously bind to the homodimer of PDGF-B, the tail sequence are brought into close proximity with their local concentration increased substantially to allow the pair of tail sequences to hybridize together with the surface-tethered DNA strands. Then the ferrocene labels of the tail sequence are drawn close to the electrode surface and produce a detectable redox current. CVs, DPVs and impedances were used to systemly characteristic this method, conforming the mechanism of the biosensor. In conclusion, this method can be implemented to the high affinity detection of PDGF-BB, dynamically increased DPV current with increasing PDGF-BB concentration ranging from 1.0 pg/mL to 20 ng/mL, with a readly achieved detection limit of 1.0 pg/mL.
(2)Whereas the aforementioned technology refered pair of proximity probes simultineouly to hybridizing with the surface-tethered DNA, it was found from the experiment that the proximity-dependent surface hybridization could not sufficiently improved the thermal stability and favorably enhanced the sensitivity. In Chapter 3, based on the mentioned mechanism of proximity-dependent surface hybridization assay, an electrochemical aptasensor based on the proximity-dependent surface hybridization assay by pair of different aptamer probes simultaneously bind to the protein for the detection of the related model protein-PDGF-BB was ulteriorly developed. The aptasensor interface was fabricated by self-assembly of a short thiolated DNA oligonucleotide 1 on the gold electrode via the alkanethiol moiety at the 5’-terminal. DNA aptamer 3 and 4 to PDGF-BB with a sequence extension at the 3’-end and the 5’-end is used as a pair of affinity probes. One aptamer probe has a electroactive ferrocene-labeled tail sequence at the 5’-end that is complementary to the surface-tethered DNA strands with a predesigned low melting temperature. Samely, the other aptamer probe with different recognization location has a tail sequence at the 3’-end that is complementary to the surface-tethered DNA strands with a predesigned low melting temperature. These two different aptamer probes could simultaneously bind to the different location of PDGF-BB homodimer. The tail sequence are brought into close proximity with their local concentration increased substantially to hybridized each other and allow the pair of tail sequences to hybridize together with the surface-tethered DNA strands. Then the ferrocene labels of the tail sequence are drawn close to the electrode surface and produce a detectable redox current. As a result, this method was proved to be wider detection range from 0.1 pg to 50 ng and lower detection limit of 0.1 pg.
(3)In chapter 4, proximity-dependent surface hybridization assay was developed to detect DNA by electrochemical biosensor method based on the high affinity and sensitivity of proximity-dependent surface hybridization assay in order to extending the application range. Different to the proteins, the nucleic acid detection based on proximity-dependent surface hybridization exploited the target DNA per se to construct a pair of proximity probes. So an oligonucleotide as the detection probe has a electroactive ferrocene-labeled tail at the 3’-end that is complementary to the target nucleic acid with a predesigned low melting temperature by 5’-end fragment. In the presense of target DNA, the detection probe and the target nucleic acid formed the hybridization complex. The hybridization one side was the detection probe that near to the other sequences of the target and make it hybridize to the surface-tethed oligonucleotide. This draw the ferrocene closed to the electrode surface and regenerated a readily detectable signal. Compare with the conventional sandwich hybridization assay, this approach ensured the ferrocene marker to enough close the electrode surface and increased the charge transport efficiency as well as improved the sensitivity remarkably. The detectable range dynamictally was from 1 fM to 1 nM and in the range the peak current exhibits a linear correlation to the log of target DNA concentration, the limit of the detection is 1 pM. This electrochemical DNA sensor can be regenerated and discriminate the mismatch with different bases sequences.
(4)In chapter 5, because of the aptamer unsatisfied the need of plentiful proteins detection, electrochemical immunosensor based-on proximity-dependent surface hybridization assay was used to detect the proteins. Herein a pair of antibody- modified oligonucleotids replaced the aptamer probes extend proximity-dependent surface hybridization assay to the proteins immunoassay that might creat a universal methodology for developing high-performance biosensors in sensitive detection of proteins. Electrochemical immunosensor based on proximity-dependent surface hybridization assay for high sensitive PSA detection was proposed. Similarly to the electrochemical aptasensor mechanism in chapter 3, a pair of mono-antibodies recognized different antigenic determinants of PSA were used to covalently crosslink two oligonucleotides. One probe has a electroactive ferrocene-labeled tail sequence at the 3’-end that is complementary to the surface-tethered DNA strands at 5’-end with a predesigned low melting temperature. Samely, the other probe has a tail sequence at the 3’-end that is complementary to the surface-tethered DNA strands at 3’-end with a predesigned low melting temperature. Furthermore, the first probe at the backward tail sequence and the seconde probe at the foreward tail sequence have a short complementary region with a low melting temperature. Antibodies simultaneously bind to the antigene, two oligonucleotides tail sequences are brought into close proximity with their local concentration increased substantially to hybridized each other and allow the pair of tail sequences to hybridize together with the surface-tethered DNA strands. Then the ferrocene labels of the tail sequence are drawn close to the electrode surface and produce a detectable redox current. The PSA was determined in the range of 25 pg to 1 ng with the detection limit of 25 pg and the immunosensor could be reusable.
(5) In chapter 6, a new sensitive, selective, reagentless and reusable electrochemical DNA sensor for detection of hybridization based on electrochemical channels switched by allosteric molecular beacon was described. This electrochemical DNA sensor employs hairpin oligonucleotide labeled with biotin as a block at one tail and labeled with NH2 at the other tail that interacted with disulfide heterocyclic compound to fix on the gold electrode surface. Because molecular beacon-like DNA stem duplex has the biggest space hindrance and the biotin as a block interacted with the 11-mercaptoundecanoic acid (MUA) increase the space hindrance, the electroactive molecules could not commendably diffuse on the electrode surface. When the probes hybridized with the target nucleic acid, the block was moved away from the electrode surface and thus formed an electrochemical pinhole channel in the 11-mercaptoundecanoic acid (MUA) self-assemble film. So the electroactive molecule can diffused, regenerated the electrochemical redox signal. The DNA target, associating with theα-thalassemia gene containing the codon 142, was determined in the range of 2.8×10-18~8.7×10-8 M, and the detection limit can be reached 1.1×10-19 M. This electrochemical sensor can be realized the high sensitive, selective, reagentless and reusable DNA biosensor and uses for indentication of SNPs.
(6) In chapter 7, a novel electrochemical immunosensor was established to detect h IgG based on gold nanoparticles tag and enhancement by surface absorbtion voltaggram assay. h IgG as the model analyte, the immunosensor interface was fabricated by immobilized GAH IgG on the glass carbon electrode surface. The immobilized antibody reacted with the analyte target-the h IgG and form sandwich configuration by using the colloidal gold-label goat anti-human immunoglobulin G (GAH IgG) as the detection probe. The size of gold nanoparticles and the apparent area of the electrode was enhanced by nanogold catalyting the reduction of HAuCl4 on the electrode surface. The self-asseble absorption of electroactive probes on the gold nanoparticles surface was exploited to measure the absorption amount on the electrode surface by DPV. The developed method is expected to hold great promise in immunosensing due to the ease of implementation, high sensitivity and specificity.
引文
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