基于量子点的核酸和蛋白质分析新技术研究
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摘要
生命组织、细菌、病毒和病菌均具有独特的核酸序列,这些特定序列的检测在卫生防疫、医学诊断、药物研究、环境科学及生物工程等领域起着重要作用。另外,生物样品中某些蛋白质浓度异常也是疾病发生的征兆,因此建立快速、准确的生物大分子的分析方法十分必要。迄今为止检测生物大分子的分析方法已有许多报道,其中,荧光分析法以其操作简单、无需底物和灵敏度高等优势,应用最为广泛。荧光分析法中常用到的荧光标记物包括有机荧光染料和新近发展的荧光纳米粒子,这其中量子点的应用发展非常迅速。相对于传统的有机染料,量子点具有一些优异的特性,使其在核酸和蛋白的分析检测中常被用作荧光探针。鉴于目前生物大分子检测的研究现状,偶合量子点优异的荧光特性,本论文发展了多种高灵敏度、高选择性的生物大分子检测新方法,主要研究内容分为三个部分：
1本部分实验发展了一种新颖的链式杂交反应模式,即以捕获探针为循环杂交反应的触发器,报告探针和目标序列之间交替互补,随后结合量子点,实现了单组分DNA的高灵敏度检测。本实验中采用报告探针分别结合捕获探针和目标序列的模式,将捕获探针和报告探针进行了特殊设计,在报告探针和目标序列、捕获探针杂交后,目标序列一端会裸露部分与捕获探针相同的碱基,从而使其它的报告探针会继续与之杂交,而体系中另外的目标序列也会再与报告探针杂交,以此循环,形成高分子量的DNA双链聚合体,建立了信号放大模式。此方法在检测DNA分子方面具有优异的性能,灵敏度高(10fM),对碱基的错配、空缺和插入的识别能力强。
在建立了链式杂交反应结合量子点检测特定序列DNA的方法后,第二节进一步将此技术拓展到蛋白质的检测。实验采用环状DNA分子作为循环杂交底物,结合适配体识别技术,建立了检测血小板衍生生长因子PDGF-BB的新方法。PDGF-BB是血清中由多种细胞所产生的能刺激平滑肌细胞、胶质等细胞增生的一种多肽,具有广泛的生理活性。PDGF-BB作为PDGF的重要亚型之一,近年来研究发现其含量对细胞转化和肿瘤生长有直接的影响,故对PDGF-BB的检测在生物学上有重要的意义。实验设计体系中两个存在互补碱基的生物素化发卡DNA分子由于其自身环的“锁”功能,不能进行杂交；而引入适配体后,与其中一条DNA分子杂交,并将其开环成为直链,引发两个DNA分子之间循环杂交。在此实验中,每个适配体分子会触发一个链式杂交反应,使大量的生物素化的DNA分子凝聚在一起,结合链霉亲和素量子点形成适配体-量子点复合物。随后将适配体-量子点复合物加入到已结合有抗原的微孔中,适配体与抗原结合,通过检测量子点的荧光强度即可确定蛋白质的含量。与非HCR模式相比,该方法信号放大效果明显,灵敏度显著提高,选择性好,并可对真实样品进行快速准确地测定。与传统的ELISA法相比,该方法更为简便可控,原材料更加稳定。
2在以上单组分DNA和蛋白质检测的基础上,进一步将方法拓展至双组分的同时测定。实验采用量子点标记寡核苷酸解链温度增强的原理,实现了双组分小RNA分子的测定。实验中对捕获探针、报告探针和目标序列在不同的结合方式下温度的影响进行了考察,结果表明在报告探针先与量子点结合后,再与目标序列和捕获探针杂交时可达到与连接酶存在时同样的效果,即增强了双链DNA的稳定性,解链温度得到提高。选择两个不同粒径的量子点标记对应的报告探针,仅需一步杂交反应即可将体系中三个部分DNA、RNA和量子点结合,无需酶的使用即可对多个组分进行同时测定,并为短链核酸的分析提供了新的思路。
为了进一步提高检测灵敏度,将层层组装的概念引入,以聚苯乙烯微球为核心,利用链霉亲和素和生物素的高度亲和性将量子点层层组装至微球表面,最后结合可与目标序列杂交的报告探针,建立了一个目标分子对应多个荧光标记物的信号放大模式。基于量子点激发光谱宽、发射光谱窄的特性,选用了两个不同发射波长的量子点,使用同一激发波长即可对双组分DNA(HIV-1,HIV-2)进行高灵敏度的检测。与未放大的方法比较,此方法灵敏度提高了100倍。
3在荧光共振能量转移体系中,为了避免光谱重叠或信号干扰,量子点常被用作供体或受体。本部分实验将量子点作为荧光共振能量转移中的供体,在夹心反应和均相体系竞争模式中,量子点均能与受体荧光染料Cy5之间通过DNA的杂交作用实现能量转移,从而对目标序列进行测定。实验首先对量子点和Cy5在载体上和均相溶液中的能量转移现象进行了考察,并进一步引入酶切循环放大的概念,采用“Y”型DNA分子结合方式,即三条DNA序列同时存在时DNA双链才能形成,并激活限制性内切酶,将其中一条分支切断,使受体与供体分离,并释放出目标序列,与其它辅助序列继续杂交,导致FRET信号的明显变化,建立了高灵敏度、高选择性的FRET-DNA检测方式。
Living organisms, bacteria, viruses and bacteria has the sequence-specific DNA or RNA, which has been became increasingly important for the diagnosis and treatment of genetic diseases, the detection of infectious agents, and forensic analysis. In addition, the concentration of some protein in blood is the indication of certain diseases. Therefore, it is highly important to establish some sensitive, accurate methods for biomacromolecules detection. Various techniques have been developed for the detection of DNA and protein. Among all of the analysis techniques, fluorescence has been widely used due to the advantages of simple operation, no substrates and the high sensitivity advantages. Semiconductor quantum dots (QDs) has been intensely developed as a new class of fluorescent label. In compare with traditional fluorescent label, QDs have unique chemical and physical properties, which accompany with recently improving understanding on QDs surface chemistry for biocompatibility and bioconjugation, lead QDs labeling to be a promising method for the detection of various biological analytes, liking DNA, protein, cells, and small organic molecules.
In consider the significance of biomacrolecules, althrough the great advances of t echno-logy on detecting biomacromolecules,the development of new method is still e xtremely significant. Herein, this subject developed several highly sensitive, specific, and novel techn-ologies by the excellent properties of QDs, which provide a new versi on for nucleic acid and protein detection. The experimental content contains three sections:
1A new quantum dot-based method to detect specific sequences of DNA has been developed. The capture and reporter probes did not hybridize to each other, but in the presence of a template they could anneal to each other via the formation of a stable ternary complex. Because of the specific design of the capture and reporter probes, the5'end of the template target DNA remained free to hybridize with another reporter. In this way, each capture DNA was an initiator strand that triggers a cascade of hybridization events between the target DNA and the reporter probe. This formed a superstructure, enhanced base stacking, and produces a strong fluorescent signal. The introduction of T4DNA ligase further stabilized the superstructure and greatly increased the fluorescence intensity, and the detection limit was as low as10fM. This fluorescence method is advantageous over conventional techniques because of its excellent ability to discriminate single base-pair mismatches and single nucleotide gap or flap.
On the basis of detecting DNA by hybridization chain reaction (HCR), we expanded this technology for the detection of PDGF-BB based on aptameric system, where stable DNA monomers assembled only upon exposure to a target PDGF-BB aptamer. In this process, two complementary stable species of biotinylated DNA hairpins coexisted in solution until the introduction of initiator aptamer strands trigger a cascade of hybridization events that yielded nicked double helices analogous to alternating copolymers. In details, the aptamer firstly opened the hairpins in the solution, created long concatamers, and then reacted with the antibody captured PDGF-BB on the well surface. Our results showed that, the coupling HCR to aptamer triggers for the amplification detection of PDGF-BB achieved a better performance in the fluorescence detection of PDGF-BB as compared to the traditional antibody-antigen-aptamer assays. The proposed method was also implemented in the analysis of human serum specimens.
2A novel multiplexed method for short RNA detection has been developed that employed a design strategy in which capture and reporter probes did not hybridize to each other and could anneal to each other in the presence of a short RNA target via the formation of a stable three-component complex. QDs functionalized with reporter DNA were used to capture a short single-stranded RNA (ssRNA) sequence from a target solution and then to specifically adsorb onto a common capture probe-modified96-well plate via a one-step template-dependent, surface hybridization for simultaneous fluorescence detection. This novel QD-based, template-dependent, surface hybridization showed several advantages compared with previous detection formats:(ⅰ) the use of reporter DNA-modified QD conjugates increased the melting temperature in comparison with conventional organic dye-modified reporter probes, leading to the detection of short RNA without the need for a ligation reaction; and (ii) QDs properties allowed multiple short RNA sequences to be simultaneously and homogeneously determined via a rapid and simple one-step hybridization, as exemplified herein.
In order to improve the sensitivity, we developed an amplification assay for multiplex detection of HIV-1and HIV-2based on QDs layer-by-layer (LBL) assembled polystyrene microsphere (PS) composite in a homogeneous format. Based on the high affinity between streptavidin and biotin, QDs were adsorbed on the surface of PS layer by layer. Biotinylated reporter then bound to the PS-QDs conjugates and was hybridized with target DNA which had been immobilized on the96-well surface. Through this way, each target DNA referred to a large number of QDs and the fluorescence signal was greatly enhanced. This PS-QDs-based sensor possesses the advantages of a simple'mix and detection'assay with enzyme-free, extremely low sample consumption, high sensitivity, and short analysis time.
3In the system of fluorescence resonance energy transfer (FRET), QDs has been always used as the donor or acceptor due to its excellent properties. In this part, we chose QDs as donor and Cy5as acceptor to form FRET for the detection of DNA. Firstly, the FRET between QDs and Cy5was investigated in a96well plate and homogeneous format respectively. Later, we used Y-shaped junction DNA which consisted of three complementary oligonucleotide branches to detect DNA. The probe was then cleaved by an endonuclease and the target was released, while the probe and the target were regenerated and attended another cleavage cycle to realize the signal amplification.
1本部分实验发展了一种新颖的链式杂交反应模式,即以捕获探针为循环杂交反应的触发器,报告探针和目标序列之间交替互补,随后结合量子点,实现了单组分DNA的高灵敏度检测。本实验中采用报告探针分别结合捕获探针和目标序列的模式,将捕获探针和报告探针进行了特殊设计,在报告探针和目标序列、捕获探针杂交后,目标序列一端会裸露部分与捕获探针相同的碱基,从而使其它的报告探针会继续与之杂交,而体系中另外的目标序列也会再与报告探针杂交,以此循环,形成高分子量的DNA双链聚合体,建立了信号放大模式。此方法在检测DNA分子方面具有优异的性能,灵敏度高(10fM),对碱基的错配、空缺和插入的识别能力强。
在建立了链式杂交反应结合量子点检测特定序列DNA的方法后,第二节进一步将此技术拓展到蛋白质的检测。实验采用环状DNA分子作为循环杂交底物,结合适配体识别技术,建立了检测血小板衍生生长因子PDGF-BB的新方法。PDGF-BB是血清中由多种细胞所产生的能刺激平滑肌细胞、胶质等细胞增生的一种多肽,具有广泛的生理活性。PDGF-BB作为PDGF的重要亚型之一,近年来研究发现其含量对细胞转化和肿瘤生长有直接的影响,故对PDGF-BB的检测在生物学上有重要的意义。实验设计体系中两个存在互补碱基的生物素化发卡DNA分子由于其自身环的“锁”功能,不能进行杂交；而引入适配体后,与其中一条DNA分子杂交,并将其开环成为直链,引发两个DNA分子之间循环杂交。在此实验中,每个适配体分子会触发一个链式杂交反应,使大量的生物素化的DNA分子凝聚在一起,结合链霉亲和素量子点形成适配体-量子点复合物。随后将适配体-量子点复合物加入到已结合有抗原的微孔中,适配体与抗原结合,通过检测量子点的荧光强度即可确定蛋白质的含量。与非HCR模式相比,该方法信号放大效果明显,灵敏度显著提高,选择性好,并可对真实样品进行快速准确地测定。与传统的ELISA法相比,该方法更为简便可控,原材料更加稳定。
2在以上单组分DNA和蛋白质检测的基础上,进一步将方法拓展至双组分的同时测定。实验采用量子点标记寡核苷酸解链温度增强的原理,实现了双组分小RNA分子的测定。实验中对捕获探针、报告探针和目标序列在不同的结合方式下温度的影响进行了考察,结果表明在报告探针先与量子点结合后,再与目标序列和捕获探针杂交时可达到与连接酶存在时同样的效果,即增强了双链DNA的稳定性,解链温度得到提高。选择两个不同粒径的量子点标记对应的报告探针,仅需一步杂交反应即可将体系中三个部分DNA、RNA和量子点结合,无需酶的使用即可对多个组分进行同时测定,并为短链核酸的分析提供了新的思路。
为了进一步提高检测灵敏度,将层层组装的概念引入,以聚苯乙烯微球为核心,利用链霉亲和素和生物素的高度亲和性将量子点层层组装至微球表面,最后结合可与目标序列杂交的报告探针,建立了一个目标分子对应多个荧光标记物的信号放大模式。基于量子点激发光谱宽、发射光谱窄的特性,选用了两个不同发射波长的量子点,使用同一激发波长即可对双组分DNA(HIV-1,HIV-2)进行高灵敏度的检测。与未放大的方法比较,此方法灵敏度提高了100倍。
3在荧光共振能量转移体系中,为了避免光谱重叠或信号干扰,量子点常被用作供体或受体。本部分实验将量子点作为荧光共振能量转移中的供体,在夹心反应和均相体系竞争模式中,量子点均能与受体荧光染料Cy5之间通过DNA的杂交作用实现能量转移,从而对目标序列进行测定。实验首先对量子点和Cy5在载体上和均相溶液中的能量转移现象进行了考察,并进一步引入酶切循环放大的概念,采用“Y”型DNA分子结合方式,即三条DNA序列同时存在时DNA双链才能形成,并激活限制性内切酶,将其中一条分支切断,使受体与供体分离,并释放出目标序列,与其它辅助序列继续杂交,导致FRET信号的明显变化,建立了高灵敏度、高选择性的FRET-DNA检测方式。
Living organisms, bacteria, viruses and bacteria has the sequence-specific DNA or RNA, which has been became increasingly important for the diagnosis and treatment of genetic diseases, the detection of infectious agents, and forensic analysis. In addition, the concentration of some protein in blood is the indication of certain diseases. Therefore, it is highly important to establish some sensitive, accurate methods for biomacromolecules detection. Various techniques have been developed for the detection of DNA and protein. Among all of the analysis techniques, fluorescence has been widely used due to the advantages of simple operation, no substrates and the high sensitivity advantages. Semiconductor quantum dots (QDs) has been intensely developed as a new class of fluorescent label. In compare with traditional fluorescent label, QDs have unique chemical and physical properties, which accompany with recently improving understanding on QDs surface chemistry for biocompatibility and bioconjugation, lead QDs labeling to be a promising method for the detection of various biological analytes, liking DNA, protein, cells, and small organic molecules.
In consider the significance of biomacrolecules, althrough the great advances of t echno-logy on detecting biomacromolecules,the development of new method is still e xtremely significant. Herein, this subject developed several highly sensitive, specific, and novel techn-ologies by the excellent properties of QDs, which provide a new versi on for nucleic acid and protein detection. The experimental content contains three sections:
1A new quantum dot-based method to detect specific sequences of DNA has been developed. The capture and reporter probes did not hybridize to each other, but in the presence of a template they could anneal to each other via the formation of a stable ternary complex. Because of the specific design of the capture and reporter probes, the5'end of the template target DNA remained free to hybridize with another reporter. In this way, each capture DNA was an initiator strand that triggers a cascade of hybridization events between the target DNA and the reporter probe. This formed a superstructure, enhanced base stacking, and produces a strong fluorescent signal. The introduction of T4DNA ligase further stabilized the superstructure and greatly increased the fluorescence intensity, and the detection limit was as low as10fM. This fluorescence method is advantageous over conventional techniques because of its excellent ability to discriminate single base-pair mismatches and single nucleotide gap or flap.
On the basis of detecting DNA by hybridization chain reaction (HCR), we expanded this technology for the detection of PDGF-BB based on aptameric system, where stable DNA monomers assembled only upon exposure to a target PDGF-BB aptamer. In this process, two complementary stable species of biotinylated DNA hairpins coexisted in solution until the introduction of initiator aptamer strands trigger a cascade of hybridization events that yielded nicked double helices analogous to alternating copolymers. In details, the aptamer firstly opened the hairpins in the solution, created long concatamers, and then reacted with the antibody captured PDGF-BB on the well surface. Our results showed that, the coupling HCR to aptamer triggers for the amplification detection of PDGF-BB achieved a better performance in the fluorescence detection of PDGF-BB as compared to the traditional antibody-antigen-aptamer assays. The proposed method was also implemented in the analysis of human serum specimens.
2A novel multiplexed method for short RNA detection has been developed that employed a design strategy in which capture and reporter probes did not hybridize to each other and could anneal to each other in the presence of a short RNA target via the formation of a stable three-component complex. QDs functionalized with reporter DNA were used to capture a short single-stranded RNA (ssRNA) sequence from a target solution and then to specifically adsorb onto a common capture probe-modified96-well plate via a one-step template-dependent, surface hybridization for simultaneous fluorescence detection. This novel QD-based, template-dependent, surface hybridization showed several advantages compared with previous detection formats:(ⅰ) the use of reporter DNA-modified QD conjugates increased the melting temperature in comparison with conventional organic dye-modified reporter probes, leading to the detection of short RNA without the need for a ligation reaction; and (ii) QDs properties allowed multiple short RNA sequences to be simultaneously and homogeneously determined via a rapid and simple one-step hybridization, as exemplified herein.
In order to improve the sensitivity, we developed an amplification assay for multiplex detection of HIV-1and HIV-2based on QDs layer-by-layer (LBL) assembled polystyrene microsphere (PS) composite in a homogeneous format. Based on the high affinity between streptavidin and biotin, QDs were adsorbed on the surface of PS layer by layer. Biotinylated reporter then bound to the PS-QDs conjugates and was hybridized with target DNA which had been immobilized on the96-well surface. Through this way, each target DNA referred to a large number of QDs and the fluorescence signal was greatly enhanced. This PS-QDs-based sensor possesses the advantages of a simple'mix and detection'assay with enzyme-free, extremely low sample consumption, high sensitivity, and short analysis time.
3In the system of fluorescence resonance energy transfer (FRET), QDs has been always used as the donor or acceptor due to its excellent properties. In this part, we chose QDs as donor and Cy5as acceptor to form FRET for the detection of DNA. Firstly, the FRET between QDs and Cy5was investigated in a96well plate and homogeneous format respectively. Later, we used Y-shaped junction DNA which consisted of three complementary oligonucleotide branches to detect DNA. The probe was then cleaved by an endonuclease and the target was released, while the probe and the target were regenerated and attended another cleavage cycle to realize the signal amplification.
引文
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