温敏性凝胶模板组装的SERS基底动“gap”研究
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摘要
表面增强拉曼光谱(SERS)技术发展至今已经能够实现单分子水平上的检测。大量研究表明这种信号的增强主要是由于贵金属纳米粒子之间的等离子耦合导致电磁场增大所致,所以SERS技术的发展强烈依赖于新型SERS活性基底设计与制备,特别是对SERS基底中贵金属纳米粒子的间隙即“gap”的调控。实际上,构建均一的、有效的SERS基底仍然具有挑战性,因为这一般都需要高成本的消耗和复杂的合成过程。采用一种简单的方法,合成能将SERS信号强烈放大并且更加均一有效的SERS基底显得尤为必要,从而有别于现有的合成方法：金属纳米粒子一旦被合成或组装好,纳米粒子的间隙就被固定。特别是,基底中纳米粒子间隙能够进行调控并且形成最佳的分布区间,即“热点”多并且分布范围窄。水凝胶聚异丙基丙烯酰胺(PNIPAM)因为其含有亲水的酰胺键和疏水的烷基链而显示出良好的温度敏感性已经被广泛研究。其在纯水中的临界相转变温度(LCST)一般为32℃,这样的温度在实际的实验中很容易操作。当体系温度低于LCST时,凝胶中的酰胺键结合大量的水分子而显示其膨胀状态；当体系温度高于LCST时,酰胺键与水分子之间的氢键会断裂,使得水分子排出凝胶网络结构,凝胶体积产生明显的收缩；而且这一膨胀收缩过程随温度的改变呈现良好的可逆性。在本论文研究中,我们将以PNIPAM为模板,对不同结构和形貌的贵金属纳米粒子进行组装,形成的复合物SERS基底的整个尺寸和金属纳米粒子间隙能够随温度变化而进行调控。从而实现对温敏性凝胶模板组装的SERS基底动“gap”研究。即在凝胶模板膨胀的状态下,将不同形貌的Au或者Ag纳米粒子组装到模板表面或者内部,此时贵金属纳米粒子间隙较大,形成的“热点”不明显,而此时将待测物分子与基底混合,分子能有效的进入到贵金属纳米粒子间隙或者周围；SERS检测时,提高温度,凝胶模板收缩,贵金属纳米粒子间隙大幅度减小,不仅使基底的“热点”瞬间增加,而且整个基底的“热点”均一性得到了提高,从而实现对待测分子的高灵敏度和高重现性SERS的检测。本论文工作包括以下几个部分：
(1)理论分析温敏性的聚合物凝胶聚异丙基丙烯酰胺(PNIPAM)的自由基聚合过程及原理,为合成合适的凝胶模板提供理论依据和指导：包括温度的控制、交联剂的数量、引发剂的种类以及凝胶膨胀收缩的机理等；同时分析各种单体的Raman信号及复合物基底的SERS信号,为待测物的SERS检测提供对照,排除对后续物检测带来的可能干扰。
(2)利用阳离子引发剂得到表面带正电荷的凝胶纳米球为模板,将L-抗坏血酸还原的Ag纳米粒子组装到凝胶模板PNIPAM纳米球表面,得到PNIPAM/Ag复合物SERS基底。提高温度,复合物的最大收缩比为18%,Ag纳米粒子间隙由30.4nm缩小至5nm以下,这一过程使基底的均一性提高,“热点”数大幅度增加,探针分子结晶紫(CV)和罗丹明6G(R6G)的SERS信号均提高了3个数量级,与用离散偶极近似法(DDA)理论模拟结果相一致。结果显示,所得的复合物基底由于温敏性凝胶模板的收缩与膨胀,使得其表面组装的贵金属纳米粒子呈现了可逆的近场耦合现象,基底获得的待测分子的SERS信号强度也是调控的。
(3)利用相似相溶原理在贵金属Au纳米粒子表面形成PNIPAM凝胶层。以该Au纳米粒子为种子进行原位再生长,得到的较大尺寸Au颗粒对加入的银离子溶液有强的静电引力作用；再采用原位还原的方法,使得大量的Ag纳米粒子被修饰生长在凝胶多孔的网络中,从而得到了卫星式结构的Au@PNIPAM/Ag复合物SERS基底。该基底中凝胶模板由湿态收缩至干态,纳米粒子间隙减小,基底均一性提高,SERS“热点”增多。值得一提的是,单个的Au@PNIPAM/Ag复合物为近微米尺寸,可以实现拉曼光学显微镜直接可视化“找点”,使得信号的重现性提高,检测结果表明对4-ATP和杀螟硫磷的SERS检测的RSD值均小于15%。
(4)利用阴离子引发剂得到表面带负电荷的凝胶纳米球为模板,将十六烷基三甲基溴化铵(CTAB)稳定的带正电荷、不同长径比的Au纳米棒组装到凝胶模板PNIPAM纳米球表面,得到PNIPAM/Au复合物SERS基底。基于同样的原理,提高温度,凝胶模板收缩,复合物中对应的纳米棒的的紫外吸收峰发生红移。采用不同波长的激发光照射复合物基底,结果表明随着温度的调节(至大于LCST),所得的探针分子的SERS信号均增强,但是,用785nm波长激发光测得的探针分子SERS信号增强最大。说明温度的升高不仅使“热点”增多,也使基底的纳米棒的表面等离子体与入射光产生共振,从而实现最佳匹配。研究表明该基底在785nm入射光激发下,实现了对10-9M农药福美双的高灵敏检测。该基底有望用于便携式拉曼光谱仪(入射光波长大都为785nm)现场检测蔬菜水果中农药残留。
With the development of Surface-enhanced Raman Spectroscopy (SERS) technology, signals can be detected with the sensitivity at the level of signal-molecule level. It is believed that one of the reasons is owing to the enormous electromagnetic enhancement of the plasmonic coupling effect among metal nanoparticles. So the progress of SERS technique is largely dependent on the development of preparation techniques of new SERS active substrates, especially for the controllable "gap" between metal nanoparticles of SERS substrates. In fact, challenges are still remained for fabricating of uniform and efficient SERS substrates. It is necessary to develop simple methods to fabricate SERS substrates which can provide high signal enhancement and uniform signals. Importantly, it is required to optimize the Raman enhancement factor and control the nano-gaps easily. In most cases, once metal nanoparticles assemblies were formed, the spatial distributions of metal nanoparticles building blocks within the composite substrate have been fixed. So, it is very important to develop a new method for more effective SERS substrates.
Poly (Nisopropylacrylamide)(PNIPAM) has been extensively studied in regard to its well-known phase behavior in aqueous solutions which has the sharpest transition among the class of thermosensitive alkylacrylamide polymers. Indeed, it undergoes a reversible phase transition at a lower critical solution temperature (LCST) of about32℃in pure water from a swollen state to a shrunken state by increasing temperature due to dissociation of the hydrophobic interaction between NIPAM segments and water. Below the LCST, PNIPAM is hydrated and the chains are in an extended conformational state. Above the LCST, PNIPAM is in a collapsed conformational state due to the breaking down of the delicate hydrophilic/hydrophobic balance in the network structure. Dehydration takes place in the PNIPAM which results in the subsequent aggregation of the PNIPAM chain and leads to the shrinking of the hydrogel. In this thesis, we will use thermo-sensitive template to assemble responsive Ag or Au nanoparticles, in which the overall composite dimensions and interparticle spatial distances can respond and adapt to external temperature stimulus. Au or Ag nanoparticles with different shapes were assembled on or inside the swollen hydrogel template, where the gaps between nanoparticles were so big that "hot spots" of these substrates were not obvious. While, if the probe molecules were mixed with these substrates, they would enter the gaps between nanoparticles. Upon SERS detection, the template can shrink due to temperature increase and this will make the gaps between nanoparticles decreasing significantly. Not only the "hot spots" on the substrates will increase instantly, but also the uniform of the whole substrate can also be improved. High sensitivity and reproducibility SERS signals can thus be obtained. This thesis work includes the followings:
(1) At first, the processes and principles of free radical polymerization for thermo-sensitive microgel polymer PNIPAM were introduced. We give the explanation for the factors affecting synthesis of the optimal microgel template, such as the temperature, the number of the cross-linking agent, kinds of initiators and the reason for the microgel swelling and shrinking et al. At the same time, the Raman spectra of all mononers and SERS spectra of the composite substrates were measured, which are served as blank control to study SERS signals of the probe molecules.
(2) PNIPAM/Ag composite substrates were synthesized through decorating Ag nanoparticles on the PNIPAM template. PNIPAM nanosphere templates with a positive surface charge were grown using a cationic initiator (AAPH). Ascorbic acid (AA)-reduced silver colloid with negative surface charge was assembled on the surface of the template. With increase of temperature, the largest shrinking ration of the composite was achieved at18%. Dynamic gaps from30.4nm to less than5nm were obtained. These changes led to an increasing of the uniformity and "hot spots" of the substrate. The SERS enhancement for multiple of CV and R6G molecules was about1000times with the template shrinking, consistent with the results estimated by DDA theoretical computation. These novel stimuli-responsive systems offer obvious advantage:a reversible near-field coupling between the Ag nanoparticles, depending on the external temperature and leading to a control over the SERS intensities of probes adsorbed on the substrate.
(3) Based on the principle of the dissolution in the similar material structure, Au core was covered by PNIPAM shell and then its growth can be controlled in situ. The obtained bigger size Au core would have strong electrostatic attraction to the Ag+(H2O)n (n=1-4) in aqueous solution. This would be followed by the reductive reaction and further growth of vast Ag+(H2O)n attracted into porosity of the gel network. Then, individual Au@PNIPAM/Ag composite with core-satellite structure which can generate plasmon resonance was obtained. Similar to the study in the third chapter, the gaps between metal nanoparticls can decrease because of the PNIPAM template shrinking from wet to dry state. Especially this kind of individual Au@PNIPAM/Ag composite can be found through Raman optical microscope. Uncertain effects on SERS signals resulting from variability of the configurations are minimized because these individual substrates are uniform relatively. The individual substrate can also be used for inspecting pesticide residues accurately and rapidly. The RSD of4-ATP and Sumithion SERS signals were all less than15%.
(4) PNIPAM nanosphere templates with a negative surface charge were grown using an anionic initiator (KPS). Au nanorods stabilized by CTAB with positive surface charge with different aspect ratio (AR) values were decorated on the thermo-sensitive hydrogel template because of electrostatic attractive force. The surface plasmon bands of Au nanorods were observed an obvious red-shift with increasing temperature due to strong plasmon coupling. This clearly demonstrated that the shrinking of the template drives the decorated Au nanorods closer to each other. Moreover, when the laser was785nm, the SERS enhancement for multiple of the same molecule was the strongest among the different lasers were used. This demonstrated that not only "hot spots" were increased but also SPR of Au nanorods matched more with the laser because of the shrinking of the template. The results showed that Thiram can be detected accurately in10-9M level. It is therefore promising to apply the PNIPAM/Au nanorods substates in protable Raman instrument (the laser is generally785nm) and detect the residual pesticide on the vegetables or apples.
(1)理论分析温敏性的聚合物凝胶聚异丙基丙烯酰胺(PNIPAM)的自由基聚合过程及原理,为合成合适的凝胶模板提供理论依据和指导：包括温度的控制、交联剂的数量、引发剂的种类以及凝胶膨胀收缩的机理等；同时分析各种单体的Raman信号及复合物基底的SERS信号,为待测物的SERS检测提供对照,排除对后续物检测带来的可能干扰。
(2)利用阳离子引发剂得到表面带正电荷的凝胶纳米球为模板,将L-抗坏血酸还原的Ag纳米粒子组装到凝胶模板PNIPAM纳米球表面,得到PNIPAM/Ag复合物SERS基底。提高温度,复合物的最大收缩比为18%,Ag纳米粒子间隙由30.4nm缩小至5nm以下,这一过程使基底的均一性提高,“热点”数大幅度增加,探针分子结晶紫(CV)和罗丹明6G(R6G)的SERS信号均提高了3个数量级,与用离散偶极近似法(DDA)理论模拟结果相一致。结果显示,所得的复合物基底由于温敏性凝胶模板的收缩与膨胀,使得其表面组装的贵金属纳米粒子呈现了可逆的近场耦合现象,基底获得的待测分子的SERS信号强度也是调控的。
(3)利用相似相溶原理在贵金属Au纳米粒子表面形成PNIPAM凝胶层。以该Au纳米粒子为种子进行原位再生长,得到的较大尺寸Au颗粒对加入的银离子溶液有强的静电引力作用；再采用原位还原的方法,使得大量的Ag纳米粒子被修饰生长在凝胶多孔的网络中,从而得到了卫星式结构的Au@PNIPAM/Ag复合物SERS基底。该基底中凝胶模板由湿态收缩至干态,纳米粒子间隙减小,基底均一性提高,SERS“热点”增多。值得一提的是,单个的Au@PNIPAM/Ag复合物为近微米尺寸,可以实现拉曼光学显微镜直接可视化“找点”,使得信号的重现性提高,检测结果表明对4-ATP和杀螟硫磷的SERS检测的RSD值均小于15%。
(4)利用阴离子引发剂得到表面带负电荷的凝胶纳米球为模板,将十六烷基三甲基溴化铵(CTAB)稳定的带正电荷、不同长径比的Au纳米棒组装到凝胶模板PNIPAM纳米球表面,得到PNIPAM/Au复合物SERS基底。基于同样的原理,提高温度,凝胶模板收缩,复合物中对应的纳米棒的的紫外吸收峰发生红移。采用不同波长的激发光照射复合物基底,结果表明随着温度的调节(至大于LCST),所得的探针分子的SERS信号均增强,但是,用785nm波长激发光测得的探针分子SERS信号增强最大。说明温度的升高不仅使“热点”增多,也使基底的纳米棒的表面等离子体与入射光产生共振,从而实现最佳匹配。研究表明该基底在785nm入射光激发下,实现了对10-9M农药福美双的高灵敏检测。该基底有望用于便携式拉曼光谱仪(入射光波长大都为785nm)现场检测蔬菜水果中农药残留。
With the development of Surface-enhanced Raman Spectroscopy (SERS) technology, signals can be detected with the sensitivity at the level of signal-molecule level. It is believed that one of the reasons is owing to the enormous electromagnetic enhancement of the plasmonic coupling effect among metal nanoparticles. So the progress of SERS technique is largely dependent on the development of preparation techniques of new SERS active substrates, especially for the controllable "gap" between metal nanoparticles of SERS substrates. In fact, challenges are still remained for fabricating of uniform and efficient SERS substrates. It is necessary to develop simple methods to fabricate SERS substrates which can provide high signal enhancement and uniform signals. Importantly, it is required to optimize the Raman enhancement factor and control the nano-gaps easily. In most cases, once metal nanoparticles assemblies were formed, the spatial distributions of metal nanoparticles building blocks within the composite substrate have been fixed. So, it is very important to develop a new method for more effective SERS substrates.
Poly (Nisopropylacrylamide)(PNIPAM) has been extensively studied in regard to its well-known phase behavior in aqueous solutions which has the sharpest transition among the class of thermosensitive alkylacrylamide polymers. Indeed, it undergoes a reversible phase transition at a lower critical solution temperature (LCST) of about32℃in pure water from a swollen state to a shrunken state by increasing temperature due to dissociation of the hydrophobic interaction between NIPAM segments and water. Below the LCST, PNIPAM is hydrated and the chains are in an extended conformational state. Above the LCST, PNIPAM is in a collapsed conformational state due to the breaking down of the delicate hydrophilic/hydrophobic balance in the network structure. Dehydration takes place in the PNIPAM which results in the subsequent aggregation of the PNIPAM chain and leads to the shrinking of the hydrogel. In this thesis, we will use thermo-sensitive template to assemble responsive Ag or Au nanoparticles, in which the overall composite dimensions and interparticle spatial distances can respond and adapt to external temperature stimulus. Au or Ag nanoparticles with different shapes were assembled on or inside the swollen hydrogel template, where the gaps between nanoparticles were so big that "hot spots" of these substrates were not obvious. While, if the probe molecules were mixed with these substrates, they would enter the gaps between nanoparticles. Upon SERS detection, the template can shrink due to temperature increase and this will make the gaps between nanoparticles decreasing significantly. Not only the "hot spots" on the substrates will increase instantly, but also the uniform of the whole substrate can also be improved. High sensitivity and reproducibility SERS signals can thus be obtained. This thesis work includes the followings:
(1) At first, the processes and principles of free radical polymerization for thermo-sensitive microgel polymer PNIPAM were introduced. We give the explanation for the factors affecting synthesis of the optimal microgel template, such as the temperature, the number of the cross-linking agent, kinds of initiators and the reason for the microgel swelling and shrinking et al. At the same time, the Raman spectra of all mononers and SERS spectra of the composite substrates were measured, which are served as blank control to study SERS signals of the probe molecules.
(2) PNIPAM/Ag composite substrates were synthesized through decorating Ag nanoparticles on the PNIPAM template. PNIPAM nanosphere templates with a positive surface charge were grown using a cationic initiator (AAPH). Ascorbic acid (AA)-reduced silver colloid with negative surface charge was assembled on the surface of the template. With increase of temperature, the largest shrinking ration of the composite was achieved at18%. Dynamic gaps from30.4nm to less than5nm were obtained. These changes led to an increasing of the uniformity and "hot spots" of the substrate. The SERS enhancement for multiple of CV and R6G molecules was about1000times with the template shrinking, consistent with the results estimated by DDA theoretical computation. These novel stimuli-responsive systems offer obvious advantage:a reversible near-field coupling between the Ag nanoparticles, depending on the external temperature and leading to a control over the SERS intensities of probes adsorbed on the substrate.
(3) Based on the principle of the dissolution in the similar material structure, Au core was covered by PNIPAM shell and then its growth can be controlled in situ. The obtained bigger size Au core would have strong electrostatic attraction to the Ag+(H2O)n (n=1-4) in aqueous solution. This would be followed by the reductive reaction and further growth of vast Ag+(H2O)n attracted into porosity of the gel network. Then, individual Au@PNIPAM/Ag composite with core-satellite structure which can generate plasmon resonance was obtained. Similar to the study in the third chapter, the gaps between metal nanoparticls can decrease because of the PNIPAM template shrinking from wet to dry state. Especially this kind of individual Au@PNIPAM/Ag composite can be found through Raman optical microscope. Uncertain effects on SERS signals resulting from variability of the configurations are minimized because these individual substrates are uniform relatively. The individual substrate can also be used for inspecting pesticide residues accurately and rapidly. The RSD of4-ATP and Sumithion SERS signals were all less than15%.
(4) PNIPAM nanosphere templates with a negative surface charge were grown using an anionic initiator (KPS). Au nanorods stabilized by CTAB with positive surface charge with different aspect ratio (AR) values were decorated on the thermo-sensitive hydrogel template because of electrostatic attractive force. The surface plasmon bands of Au nanorods were observed an obvious red-shift with increasing temperature due to strong plasmon coupling. This clearly demonstrated that the shrinking of the template drives the decorated Au nanorods closer to each other. Moreover, when the laser was785nm, the SERS enhancement for multiple of the same molecule was the strongest among the different lasers were used. This demonstrated that not only "hot spots" were increased but also SPR of Au nanorods matched more with the laser because of the shrinking of the template. The results showed that Thiram can be detected accurately in10-9M level. It is therefore promising to apply the PNIPAM/Au nanorods substates in protable Raman instrument (the laser is generally785nm) and detect the residual pesticide on the vegetables or apples.
引文
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