新型生物传感技术用于核酸和蛋白质的检测
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摘要
蛋白质和核酸是生命体中最重要的两类生物大分子,对人类的遗传疾病和新陈代谢有重要影响,因此建立简单、灵敏、高效的蛋白质、核酸分析方法对临床诊断、药物筛选等医学研究具有重要的意义。针对当前对蛋白质和核酸检测技术提出的高灵敏、高选择、高通量、高特异性和操作简便的要求,本论文建立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)本章建立了一种用于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5’末端修饰二茂铁,3’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,并使末端标记的二茂铁与电极充分接近,从而产生显著的氧化还原电流。结果表明,该方法的检测线性范围宽,为1 fM到1 nM,而且能够成功地识别不同碱基数错配的序列。与传统夹心式杂交分析相比,该法保证了末端标记的二茂铁与电极充分接近,电子转移效率提高,灵敏度得到了显著改善。而且由于邻近分析使用了更短的互补序列,有效提高了其选择性。
(2)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用于检测模型分析物―血小板源性生长因子BB(PDGF-BB)的表面邻近杂交分析电化学适体传感方法。该方法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,使得适体探针末端标记的二茂铁与电极充分接近而发生高效电子传递,从而产生显著的氧化还原电流。该法可实现对PDGF-BB的高特异性地检测,动态响应范围可从1.0 pg/mL到20 ng/mL,检测限可达1.0 pg/mL。
(3)构建了一种脂质体滚环扩增免疫传感技术用于超灵敏地检测目标蛋白PSA,通过在脂质体中包埋引物探针和滚环扩增反应两步放大,使得灵敏度大大提高,显示了很好的线性范围和重现性,高选择性,检测范围从0.1 fg/mL到0.1 ng/mL,使检测下限大大降低,达到0.08 fg/mL。
Protein and nucleic acid are the most two important macro-biomolecules, playing essential roles in the fields of genetic disease and metabolism. Therefore, it is highly significant that constructing simple, sensitive and efficient analytical methods for detecting protein and nucleic acid in the area of clinical diagnosis and drug screening. To satisfy and achieve the demand of the simple, high sensitivity, selectivity and affinity, and high throughout for the detection of proteins and DNA, we develop a series of novel electrochemical biosensor methods and provide a high performance platform. These results primarily proved that the proposed technology is reasonably comparable with the traditional detection methods, indicating the practicability of using the proposed method in clinical diagnosis. The detailed content described as follows:
(1) We develop a novel electrochemical DNA (E-DNA) biosensor for simple, rapid, and specific detection of nucleic acids based on the proximity-dependent surface hybridization assay in order to widening the application area. This E-DNA biosensor was constructed by self-assembly of a 3’short thiolated capture probe on the gold electrode. DNA detection was realized by outputting a remarkable redox current of 5’ferrocene (Fc) tail labeled probe. When the target DNA was introduced into the system, it was complementary to the 5’Fc labeled probe at the one half-segment and complementary to the 5’short thiolated capture probe at the other half-segment, resulting in forming a stable duplex complex. As a result, the Fc probe was proximate to the electrode surface and the Faradaic current was observed. This E-DNA biosensor was proved to have a low detection limit (1 fM) and a wide dynamic range (from 1 fM to 1 nM) due to the stable hybridization mode. In addition, the sensing system could discriminate the complementary sequence from mismatch sequences, with high sensitivity, stability, and reusability. Compared 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.
(2) We develop 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 simultaneously recognize the target molecules and form the proximity probes so that some complementary sequence hybridization enhanced the stability. When aptamer pairs simultaneously binding to the homodimer of PDGF-BB, 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. 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 the detection limit of 1.0 pg/mL.
(3) A novel rolling circle amplification (RCA) immunoassay based on DNA-encapsulating liposomes, liposome-RCA immunoassay, was developed for ultrasensitive protein detection. This technique utilized antibody-modified liposomes with DNA prime probes encapsulated and a linear RCA reaction for the determination of prostate-specific antigen (PSA),. The results revealed that the technique exhibited a dynamic response to PSA over a six-decade concentration range from 0.1 fg mL-1 to 0.1 ng mL-1 with a limit of detection as low as 0.08 fg mL-1 and a high dose-response sensitivity.
(1)本章建立了一种用于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5’末端修饰二茂铁,3’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,并使末端标记的二茂铁与电极充分接近,从而产生显著的氧化还原电流。结果表明,该方法的检测线性范围宽,为1 fM到1 nM,而且能够成功地识别不同碱基数错配的序列。与传统夹心式杂交分析相比,该法保证了末端标记的二茂铁与电极充分接近,电子转移效率提高,灵敏度得到了显著改善。而且由于邻近分析使用了更短的互补序列,有效提高了其选择性。
(2)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用于检测模型分析物―血小板源性生长因子BB(PDGF-BB)的表面邻近杂交分析电化学适体传感方法。该方法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,使得适体探针末端标记的二茂铁与电极充分接近而发生高效电子传递,从而产生显著的氧化还原电流。该法可实现对PDGF-BB的高特异性地检测,动态响应范围可从1.0 pg/mL到20 ng/mL,检测限可达1.0 pg/mL。
(3)构建了一种脂质体滚环扩增免疫传感技术用于超灵敏地检测目标蛋白PSA,通过在脂质体中包埋引物探针和滚环扩增反应两步放大,使得灵敏度大大提高,显示了很好的线性范围和重现性,高选择性,检测范围从0.1 fg/mL到0.1 ng/mL,使检测下限大大降低,达到0.08 fg/mL。
Protein and nucleic acid are the most two important macro-biomolecules, playing essential roles in the fields of genetic disease and metabolism. Therefore, it is highly significant that constructing simple, sensitive and efficient analytical methods for detecting protein and nucleic acid in the area of clinical diagnosis and drug screening. To satisfy and achieve the demand of the simple, high sensitivity, selectivity and affinity, and high throughout for the detection of proteins and DNA, we develop a series of novel electrochemical biosensor methods and provide a high performance platform. These results primarily proved that the proposed technology is reasonably comparable with the traditional detection methods, indicating the practicability of using the proposed method in clinical diagnosis. The detailed content described as follows:
(1) We develop a novel electrochemical DNA (E-DNA) biosensor for simple, rapid, and specific detection of nucleic acids based on the proximity-dependent surface hybridization assay in order to widening the application area. This E-DNA biosensor was constructed by self-assembly of a 3’short thiolated capture probe on the gold electrode. DNA detection was realized by outputting a remarkable redox current of 5’ferrocene (Fc) tail labeled probe. When the target DNA was introduced into the system, it was complementary to the 5’Fc labeled probe at the one half-segment and complementary to the 5’short thiolated capture probe at the other half-segment, resulting in forming a stable duplex complex. As a result, the Fc probe was proximate to the electrode surface and the Faradaic current was observed. This E-DNA biosensor was proved to have a low detection limit (1 fM) and a wide dynamic range (from 1 fM to 1 nM) due to the stable hybridization mode. In addition, the sensing system could discriminate the complementary sequence from mismatch sequences, with high sensitivity, stability, and reusability. Compared 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.
(2) We develop 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 simultaneously recognize the target molecules and form the proximity probes so that some complementary sequence hybridization enhanced the stability. When aptamer pairs simultaneously binding to the homodimer of PDGF-BB, 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. 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 the detection limit of 1.0 pg/mL.
(3) A novel rolling circle amplification (RCA) immunoassay based on DNA-encapsulating liposomes, liposome-RCA immunoassay, was developed for ultrasensitive protein detection. This technique utilized antibody-modified liposomes with DNA prime probes encapsulated and a linear RCA reaction for the determination of prostate-specific antigen (PSA),. The results revealed that the technique exhibited a dynamic response to PSA over a six-decade concentration range from 0.1 fg mL-1 to 0.1 ng mL-1 with a limit of detection as low as 0.08 fg mL-1 and a high dose-response sensitivity.
引文
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