自组装纳米四面体手性机制及高灵敏生物传感检测
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摘要
本论文以多种纳米颗粒材料为构成元素并结合DNA自组装技术，实现了三维空间手性纳米颗粒四面体的可控组装；利用DNA分子的稳定性、可编程性以及空间四面体的多元素优势，通过改变四面体的空间构象建立了一系列简单、快速、超灵敏的生物传感检测新方法。
首先，制备了五种不同的纳米颗粒，分别是10nm，15nm和25nm的金纳米颗粒、10nm的银纳米颗粒以及5nm的半导体荧光量子点，以上五种纳米材料形貌均一，分散性好且在水溶液中稳定。空间四面体是由四条末端修饰巯基或者氨基的单链DNA通过杂交互补形成的。纳米颗粒通过与DNA末端修饰的巯基或者氨基偶联，构成空间四面体的四个顶点。通过改变四面体顶端纳米颗粒的种类或者尺寸，成功构建了六种不同类型的三维空间四面体组装材料，这六种四面体分散性好、结构稳定，且组装产率都在75%以上。实验中运用多种表征手段对其空间结构进行了表征。圆二色光谱研究发现，除了第一类四面体外，其余五种四面体在可见光区（300-800nm）都有明显的手性信号，特别是第五种和第六种四面体呈现出几乎完全对称的手性光谱。通过对比六种不同类型四面体的空间结构发现，当四面体顶端的四个颗粒完全相同时（类型一），四面体是没有手性信号的。随着不同纳米颗粒的加入，四面体的手性信号逐渐增加，其中第五种和第六种四面体的手性信号最强。对于四面体的手性来源，可以从以下几个方面解释：1）不同纳米颗粒的加入打破了四面体的对称骨架；2）不同颗粒之间的等离子共振相互作用；3）纳米颗粒之间的DNA手性分子的手性转移作用。
其次，分别用DNA和氯化钠（NaCl）两种不同圆二色性的分子成功组装了金纳米颗粒异质二聚体。基于二聚体是纳米颗粒组装体中最简单的结构，以此为代表研究其手性来源有利于观察组装体几何构型的变化与其手性信号的关系。结果显示，这两种二聚体在500nm附近都有明显的手性信号，DNA组装的二聚体的手性信号为负值，而NaCl组装的二聚体的手性信号为正值，且前者的信号强度比后者高，这与软件模拟的结果完全吻合。该结果说明手性分子不是手性产生的根本原因，二聚体的手性应该是来自本身的空间结构。实验中采用冷冻电子显微技术结合计算机软件模拟对两种二聚体的空间结构进行了研究发现，二聚体中两个颗粒之间的夹角是手性信号产生的根本原因。首次提出了纳米组装体手性起源新理论。
第三，构建了手性和非手性两种不同的空间四面体，当目标DNA存在时，两种四面体空间构象发生改变，手性信号呈现出不同的变化趋势，一种的手性信号逐渐增强而另外一种的手性信号逐渐减弱。实验中采用多种手段对两种四面体空间构象的变化做了表征。基于空间四面体的构象变化与手性信号的关系，建立了两种不同模式的DNA手性传感检测方法。在最优条件下，DNA的检测灵敏度可以达到3.4aM，该检测限远远低于其它光学传感器，从而建立了一种快速、超灵敏的DNA手性检测方法。
第四，制备高产、均一的银纳米颗粒空间四面体结构，在DNA四面体的六个边上分别嵌入核酸适配体，目标分子能与自身适配体结合形成DNA茎环结构，纳米颗粒之间产生强的“热点”区域，从而使此区域内的拉曼信标分子的信号明显增强。基于此原理，分别构建了磺胺二甲氧嘧啶(SDM)和前列腺特异性蛋白(PSA)的四面体单重拉曼超灵敏检测新方法。信标分子采用对氨基苯硫酚(4-ATP),根据信标分子的信号强度与目标分子浓度建立标准曲线，在最优条件下，SDM和PSA的检测限分别可达到86.2pM和40zM，是目前基于纳米材料传感检测的最低检测限。利用空间四面体的多元素优势，通过采用三种拉曼信标分子和三种核酸适配体，建立了三种靶蛋白浓度与SERS信号之间的线性关系并应用于癌症标志蛋白的拉曼多重检测。检测的三种目标物依次为前列腺特异性蛋白，凝血酶和粘蛋白1，对应的检测限依次为：1.2aM，158aM和26aM。相比于其它SERS传感器，该方法稳定性好、灵敏度高并且有很好的特异性。
In this paper, three-dimensional chiral nanoparticle pyramids were achieved based onDNA self-assembly technology combined with variety of nanoparticles materials; Takingadvantage of the stability and programmability of DNA and multiplexed elements of spatialpyramids, a series of new methods of simple, rapid, ultra-sensitive biosensor detection wereestablished by changing the tetrahedral conformation.
Firstly, five different nanoparticles were synthesized, which were10nm,15nm and25nm gold nanoparticles,10nm silver nanoparticles and5nm fluorescent quantum dots, thesematerials showed uniform morphology, good dispersion and stability in aqueous solution.Three-dimensional pyramids were assembled using four single-stranded DNA (ssDNA)modified with sulfhydryl or amino group, and nanoparticles were attached on each top ofpyramids by coupling with the sulfhydryl or amino group of ssDNA. By changing the types orsizes of nanoparticle attached on the top of pyramids, six different types of three-dimensionalpyramids with well dispersion and stability were constructed successfully with the yieldexceeding75%. A variety of optical instruments were used to characterize the spatial structureof pyramids. Circular dichroism spectroscopy showed that, in addition to the first class ofpyramid, the others all exhibited significant circular dichroism (CD) signals in the visibleregion (300-800nm), in particular, the fifth and the sixth pyramids emerged almostcompletely symmetric chiral spectrum. By comparing the spatial structures of six differenttypes of pyramids, we found that pyramids formed with four identical particles were achiral(the first type), with the addition of different nanoparticles, the CD intensity of nanoparticlespyramids were gradually increased, the chiral signal of the fifth and sixth pyramids were thestrongest. The origin of chirality of nanoparticle pyramids can be interpreted from thefollowing aspects:1) the addition of different nanoparticles breaks the symmetric frame ofpyramids;2) the interactions between plasmical nanoparticles with different shapes and size;3) the chirality of DNA molecules was transferred to the nanoparticles in pyramids.
Secondly, gold nanoparticles heterodimers were assembled with a chiral (DNA) and anachiral molecule (NaCl), respectively. Because the dimers are the simplest structure innanoparticle assemblies, they could be used as a respensentative material to study the originof chirality, it is easy to clearly observe the relationship between the conformational changeand the CD signal of assemblies. It is found that these two types of heterodimers both showedsignificant CD signals at the visible region of500nm, the value of CD signal from DNAassembled dimers was negative and the value of CD signal from NaCl assembled dimers waspositive, the intensity of CD signal from the former was stronger than the later，which wasagreed well with the software simulation dates. The results showed that chiral molecules werenot the origin of chirality of heterodimers and the structure of heterodimers may be the originof chirality. Cryo-electron microscopy combined with simulation software were used to studythe spatial structure of the two types of dimers, it was found that the angles between twoparticles in heterodimers was the origin of the CD signal. A new theory of the origin ofchirality in nanoparticle assemblies was first proposed.
Thirdly, two different three-dimensional pyramids (chiral and achiral) were constructed,with the addition of target DNA, the conformations of two different pyramids were changedwhich altered the CD signals of the two pyramids, one CD signal was gradually increased andthe other one was gradually decreased. Various methods were used to characterize theconformation changes of these two pyramids. Based on the relationship between theconformation change and the CD signals of pyramids, two different methods were establishedto detection of DNA molecules. Under optimal conditions, a limit of detection (LOD) forDNA was as low as3.4aM, which was much lower than other optical sensors. Here, a rapid,ultra-sensitive chiral sensor for DNA detection was established.
Lastly, uniform silver nanoparticles pyramids were prepared with high yields, and thenDNA aptamers were inserted in each side of nanoparticles pyramids, they can bind with itsaptamer and form a DNA stem-loop structure and produced a strong "hot spots" between thesilver nanoparticles, so the signal of the Raman reporter molecular existed in this region wassignificantly enhanced. Based on this principle， an ultrasensitive Raman sensor forsulfadimethoxine (SDM) and prostate-specific protein (PSA) were constructed using Ag NPspyramids, respectively, in which amino thiophenol (4-ATP) was used as Raman reportermolecule. Based on the relationship between the intensity of Raman signals and theconcentration of SDM and PSA, a standard curve for these two molecules detection has beenestablished, the LOD of SDM and PSA were82.6pM and40zM respectively, which was theultrasensitive method for SDM or PSA detection using surface-enhanced Raman scattering(SERS). Taking advantage of the multi-element of pyramids, a multiplexed Raman sensor forbio-marker proteins was constructed based on three Raman reporter molecules and threeprotein aptamers. Three targets used in this work were prostate-specific protein, thrombin, andmucin-1, the corresponding LOD were1.2aM,158aM and26aM, respectively. Comparedwith other SERS sensors, this method showed well stability and high sensitivity.
首先，制备了五种不同的纳米颗粒，分别是10nm，15nm和25nm的金纳米颗粒、10nm的银纳米颗粒以及5nm的半导体荧光量子点，以上五种纳米材料形貌均一，分散性好且在水溶液中稳定。空间四面体是由四条末端修饰巯基或者氨基的单链DNA通过杂交互补形成的。纳米颗粒通过与DNA末端修饰的巯基或者氨基偶联，构成空间四面体的四个顶点。通过改变四面体顶端纳米颗粒的种类或者尺寸，成功构建了六种不同类型的三维空间四面体组装材料，这六种四面体分散性好、结构稳定，且组装产率都在75%以上。实验中运用多种表征手段对其空间结构进行了表征。圆二色光谱研究发现，除了第一类四面体外，其余五种四面体在可见光区（300-800nm）都有明显的手性信号，特别是第五种和第六种四面体呈现出几乎完全对称的手性光谱。通过对比六种不同类型四面体的空间结构发现，当四面体顶端的四个颗粒完全相同时（类型一），四面体是没有手性信号的。随着不同纳米颗粒的加入，四面体的手性信号逐渐增加，其中第五种和第六种四面体的手性信号最强。对于四面体的手性来源，可以从以下几个方面解释：1）不同纳米颗粒的加入打破了四面体的对称骨架；2）不同颗粒之间的等离子共振相互作用；3）纳米颗粒之间的DNA手性分子的手性转移作用。
其次，分别用DNA和氯化钠（NaCl）两种不同圆二色性的分子成功组装了金纳米颗粒异质二聚体。基于二聚体是纳米颗粒组装体中最简单的结构，以此为代表研究其手性来源有利于观察组装体几何构型的变化与其手性信号的关系。结果显示，这两种二聚体在500nm附近都有明显的手性信号，DNA组装的二聚体的手性信号为负值，而NaCl组装的二聚体的手性信号为正值，且前者的信号强度比后者高，这与软件模拟的结果完全吻合。该结果说明手性分子不是手性产生的根本原因，二聚体的手性应该是来自本身的空间结构。实验中采用冷冻电子显微技术结合计算机软件模拟对两种二聚体的空间结构进行了研究发现，二聚体中两个颗粒之间的夹角是手性信号产生的根本原因。首次提出了纳米组装体手性起源新理论。
第三，构建了手性和非手性两种不同的空间四面体，当目标DNA存在时，两种四面体空间构象发生改变，手性信号呈现出不同的变化趋势，一种的手性信号逐渐增强而另外一种的手性信号逐渐减弱。实验中采用多种手段对两种四面体空间构象的变化做了表征。基于空间四面体的构象变化与手性信号的关系，建立了两种不同模式的DNA手性传感检测方法。在最优条件下，DNA的检测灵敏度可以达到3.4aM，该检测限远远低于其它光学传感器，从而建立了一种快速、超灵敏的DNA手性检测方法。
第四，制备高产、均一的银纳米颗粒空间四面体结构，在DNA四面体的六个边上分别嵌入核酸适配体，目标分子能与自身适配体结合形成DNA茎环结构，纳米颗粒之间产生强的“热点”区域，从而使此区域内的拉曼信标分子的信号明显增强。基于此原理，分别构建了磺胺二甲氧嘧啶(SDM)和前列腺特异性蛋白(PSA)的四面体单重拉曼超灵敏检测新方法。信标分子采用对氨基苯硫酚(4-ATP),根据信标分子的信号强度与目标分子浓度建立标准曲线，在最优条件下，SDM和PSA的检测限分别可达到86.2pM和40zM，是目前基于纳米材料传感检测的最低检测限。利用空间四面体的多元素优势，通过采用三种拉曼信标分子和三种核酸适配体，建立了三种靶蛋白浓度与SERS信号之间的线性关系并应用于癌症标志蛋白的拉曼多重检测。检测的三种目标物依次为前列腺特异性蛋白，凝血酶和粘蛋白1，对应的检测限依次为：1.2aM，158aM和26aM。相比于其它SERS传感器，该方法稳定性好、灵敏度高并且有很好的特异性。
In this paper, three-dimensional chiral nanoparticle pyramids were achieved based onDNA self-assembly technology combined with variety of nanoparticles materials; Takingadvantage of the stability and programmability of DNA and multiplexed elements of spatialpyramids, a series of new methods of simple, rapid, ultra-sensitive biosensor detection wereestablished by changing the tetrahedral conformation.
Firstly, five different nanoparticles were synthesized, which were10nm,15nm and25nm gold nanoparticles,10nm silver nanoparticles and5nm fluorescent quantum dots, thesematerials showed uniform morphology, good dispersion and stability in aqueous solution.Three-dimensional pyramids were assembled using four single-stranded DNA (ssDNA)modified with sulfhydryl or amino group, and nanoparticles were attached on each top ofpyramids by coupling with the sulfhydryl or amino group of ssDNA. By changing the types orsizes of nanoparticle attached on the top of pyramids, six different types of three-dimensionalpyramids with well dispersion and stability were constructed successfully with the yieldexceeding75%. A variety of optical instruments were used to characterize the spatial structureof pyramids. Circular dichroism spectroscopy showed that, in addition to the first class ofpyramid, the others all exhibited significant circular dichroism (CD) signals in the visibleregion (300-800nm), in particular, the fifth and the sixth pyramids emerged almostcompletely symmetric chiral spectrum. By comparing the spatial structures of six differenttypes of pyramids, we found that pyramids formed with four identical particles were achiral(the first type), with the addition of different nanoparticles, the CD intensity of nanoparticlespyramids were gradually increased, the chiral signal of the fifth and sixth pyramids were thestrongest. The origin of chirality of nanoparticle pyramids can be interpreted from thefollowing aspects:1) the addition of different nanoparticles breaks the symmetric frame ofpyramids;2) the interactions between plasmical nanoparticles with different shapes and size;3) the chirality of DNA molecules was transferred to the nanoparticles in pyramids.
Secondly, gold nanoparticles heterodimers were assembled with a chiral (DNA) and anachiral molecule (NaCl), respectively. Because the dimers are the simplest structure innanoparticle assemblies, they could be used as a respensentative material to study the originof chirality, it is easy to clearly observe the relationship between the conformational changeand the CD signal of assemblies. It is found that these two types of heterodimers both showedsignificant CD signals at the visible region of500nm, the value of CD signal from DNAassembled dimers was negative and the value of CD signal from NaCl assembled dimers waspositive, the intensity of CD signal from the former was stronger than the later，which wasagreed well with the software simulation dates. The results showed that chiral molecules werenot the origin of chirality of heterodimers and the structure of heterodimers may be the originof chirality. Cryo-electron microscopy combined with simulation software were used to studythe spatial structure of the two types of dimers, it was found that the angles between twoparticles in heterodimers was the origin of the CD signal. A new theory of the origin ofchirality in nanoparticle assemblies was first proposed.
Thirdly, two different three-dimensional pyramids (chiral and achiral) were constructed,with the addition of target DNA, the conformations of two different pyramids were changedwhich altered the CD signals of the two pyramids, one CD signal was gradually increased andthe other one was gradually decreased. Various methods were used to characterize theconformation changes of these two pyramids. Based on the relationship between theconformation change and the CD signals of pyramids, two different methods were establishedto detection of DNA molecules. Under optimal conditions, a limit of detection (LOD) forDNA was as low as3.4aM, which was much lower than other optical sensors. Here, a rapid,ultra-sensitive chiral sensor for DNA detection was established.
Lastly, uniform silver nanoparticles pyramids were prepared with high yields, and thenDNA aptamers were inserted in each side of nanoparticles pyramids, they can bind with itsaptamer and form a DNA stem-loop structure and produced a strong "hot spots" between thesilver nanoparticles, so the signal of the Raman reporter molecular existed in this region wassignificantly enhanced. Based on this principle， an ultrasensitive Raman sensor forsulfadimethoxine (SDM) and prostate-specific protein (PSA) were constructed using Ag NPspyramids, respectively, in which amino thiophenol (4-ATP) was used as Raman reportermolecule. Based on the relationship between the intensity of Raman signals and theconcentration of SDM and PSA, a standard curve for these two molecules detection has beenestablished, the LOD of SDM and PSA were82.6pM and40zM respectively, which was theultrasensitive method for SDM or PSA detection using surface-enhanced Raman scattering(SERS). Taking advantage of the multi-element of pyramids, a multiplexed Raman sensor forbio-marker proteins was constructed based on three Raman reporter molecules and threeprotein aptamers. Three targets used in this work were prostate-specific protein, thrombin, andmucin-1, the corresponding LOD were1.2aM,158aM and26aM, respectively. Comparedwith other SERS sensors, this method showed well stability and high sensitivity.
引文
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