点击功能化的叶酸受体靶向荧光纳米探针用于肿瘤细胞成像

采用点击化学偶联法对荧光二氧化硅纳米粒子表面进行叶酸功能化修饰,构建了一种叶酸受体靶向的荧光纳米探针,并成功用于肿瘤细胞的成像研究.首先通过St?ber法制备包裹钌联吡啶的荧光二氧化硅纳米粒子(RSiNPs),然后利用叠氮化硅烷偶联剂(Az-PTES)的水解反应在其表面引入叠氮基团,最后通过点击化学反应将炔丙基叶酸衍生物偶联到粒子表面.利用红外光谱对其偶联前后的叠氮基特征峰(2105 cm-1)进行表征,证实了叶酸功能化的荧光纳米探针(RSiNPs-Folate)已被成功制备.在生理pH条件下,以458 nm为激发波长,RSiNPs-Folate在601 nm处发射较强的红色荧光,且光稳定性较好.细胞成像结果表明,这种叶酸受体靶向的荧光纳米探针能够有效地标记叶酸受体呈阳性的人宫颈癌细胞(HeLa),而叶酸受体呈阴性的人肺癌细胞(A549)未观察到明显的荧光.叶酸竞争性结合实验证明了这种叶酸受体介导的肿瘤细胞成像机制.此探针能够实现混合细胞体系中HeLa细胞的选择性识别与荧光成像.与酰胺化反应偶联叶酸相比,这种点击功能化的纳米探针的合成方法简单、反应条件温和、产率高,可用于不同肿瘤细胞的荧光标记与成像.     

靶向性纳米金探针对宫颈癌细胞的激光扫描共聚焦散射成像

制备了粒径均一的纳米金颗粒, 再对其表面进行叶酸修饰, 制得具有靶向性的纳米金探针. 利用激光扫描共聚焦显微镜(LSCM), 对靶向性纳米金的细胞特异性散射成像进行研究. 实验结果表明, 人宫颈癌细胞(Hela)对纳米金-叶酸的摄取作用强于对纳米金的摄取, 但随着时间的延长, 两者的差别逐渐减小. 表明在适当的时间内纳米金-叶酸探针对宫颈癌细胞具有良好的靶向性.     

包裹RuBpy二氧化硅荧光纳米探针的制备及其用于肝癌细胞的识别

制备了两种不同官能团修饰的偶联亲和素的包裹钌联吡啶(RuBpy)二氧化硅荧光纳米探针A和探针B,并分别用于肝癌细胞的识别。通过反相微乳液法制备得到表面修饰不同官能团的纳米颗粒,然后通过亲和素与羧基化包裹RuBpy二氧化硅纳米颗粒相互连接而制备得到探针A;通过亲和素与PEG修饰的荧光二氧化硅纳米颗粒相互作用而制备得到探针B。与探针A不同的是,探针B通过一个长链PEG分子将亲和素与荧光二氧化硅纳米颗粒偶联在一起。利用免疫荧光成像法将这两种探针分别用于人肝癌细胞的识别,结果表明,含有长链PEG分子的探针B更能够有效地识别肝癌细胞表面肿瘤标志物癌胚抗原(CEA)。     

同步辐射X射线成像技术在纳米探针细胞成像中的应用

纳米探针在肿瘤的高灵敏成像和高效治疗可视化方面具有重要的应用前景.通过细胞原位成像技术揭示纳米探针与细胞间的相互作用将为其临床应用奠定生物学基础.同步辐射X射线成像技术是研究纳米探针细胞原位摄取、胞内代谢及构效关系的重要方法.本文系统总结了基于同步辐射光源X射线成像技术在纳米探针细胞原位成像方面的研究进展,包括纳米探针的细胞原位成像、亚细胞结构原位成像、细胞原位价态分析、细胞原位定量成像以及细胞原位三维成像.此外,本文还对可用于纳米探针细胞成像的X射线成像技术的发展趋势进行了探讨.     

基于碳点多功能纳米载药体系的制备及pH响应释药性能研究

采用一步微波法成功制备了表面带氨基的荧光纳米碳点CDots, 并通过酰胺化反应将靶向基团叶酸接枝到碳点表面, 成功获得中间产物CDots-FA. 在此基础上, 通过已合成四臂端酰肼基化合物2与抗肿瘤药物阿霉素(DOX)连接, 实现在碳点表面的阿霉素药物分子的化学键合, 最终获得多功能纳米载药体系DOX-CDots-FA. 利用原子力显微镜(AFM)、高分辨透射电镜(HR-TEM)和荧光光谱仪对荧光纳米碳点CDots的性能进行表征, 并通过核磁共振、紫外-可见吸收光谱对DOX-CDots-FA结构、接枝率进行了表征. 同时对纳米载药体系DOX-CDots-FA体外药物释放行为、细胞毒性及细胞摄取成像进行了系统的研究. 结果表明, DOX-CDots-FA具有良好的pH响应性. 叶酸靶向基团能加速DOX-CDots-FA被HeLa (FR+)细胞摄取, 并表现出更强的细胞毒性. 同时细胞摄入成像实验表明, 在叶酸靶向作用下, DOX-CDots-FA通过内吞作用进入HeLa细胞, 随后阿霉素被释放出来并进入细胞核区域, 抑制细胞的生长, 从而实现靶向治疗, 降低毒副作用.     

纳米颗粒探针检测核糖体失活蛋白

建立了纳米颗粒探针检测核糖体失活蛋白的方法.以聚苯乙烯纳米颗粒为载体并进行表面修饰,在其表面固定特异性蓖麻毒单抗并作为探针,用于蓖麻毒蛋白的检测.采用场流分离技术对纳米颗粒探针进行准确表征,并利用扫描电镜比较聚苯乙烯纳米颗粒的形貌变化.结果表明:通过扫描电镜图片能够观察到聚苯乙烯纳米颗粒表面结合的蛋白,此纳米颗粒探针能够与蓖麻毒蛋白等核糖体失活蛋白的特异性结合,应用于目标蛋白的检测.     

透明质酸-量子点的制备及应用

基于量子点(QD)独特的光学成像特性, 采用化学合成法制备了透明质酸(HA)修饰的水溶性纳米量子点(HA-QD), 并将其应用于特异性受体CD44的识别研究中. 体外细胞实验结果证实, 在透明质酸受体的介导下, 该纳米复合物可使小鼠肺腺癌细胞LA795显示特异性的荧光成像. 本研究为建立针对透明质酸受体的肿瘤活体检测及研究肿瘤的发生发展提供了重要的纳米靶向荧光探针.     

SERS标记的金纳米棒探针用于免疫检测

报道了基于金纳米棒表面增强拉曼散射(SERS)的免疫检测. 将拉曼活性分子对巯基苯甲酸吸附于金纳米棒表面, 制备出SERS标记的金纳米棒探针. 该探针和蛋白抗体结合形成SERS标记抗体. 通过SERS标记抗体、待测抗原和俘获抗体(固体基底上修饰的抗体, 即俘获抗体)之间的免疫应答反应, 将金纳米棒探针组装到固体基底上, 形成SERS标记抗体-抗原-俘获抗体 “三明治”夹心复合体. 待测抗原浓度越大, 固体基底上俘获的金纳米棒探针的数目越多, 从而可通过SERS信号的强弱来检测待测抗原的浓度. 由于金纳米棒的表面等离子体共振(SPR)峰位置可以在较宽的范围内调控, 可通过激发光和SPR的耦合来提高SERS信号, 从而提高免疫检测的灵敏度. 单组分抗原可检出的浓度范围高于1×10－8 mg/mL.     

靶向髓过氧化酶的双模态探针分子的合成与表征

5-羟色胺(5-HT)对动脉粥样硬化斑块中巨噬细胞分泌的髓过氧化酶(MPO)具有靶向功能,通过酯化反应将5-HT接枝到两亲型聚合物聚衣康酸接枝聚乙二醇接枝十二胺(PIA-g-PEG-g-DDA)上,制备了具有靶向功能的两亲型聚合物(5-HT-g-PIA-g-PEG-g-DDA).采用配体交换法,将超顺磁性氧化铁纳米粒子(SPION)包覆在该聚合物上制备具有磁性的纳米粒子(5-HT-g-PIA-g-PEG-g-DDA@SPION),通过静电力结合使其与碳点(CDs)结合,制备具有荧光成像和核磁造影成像的双模态探针的纳米粒子.采用动态光散射(DLS)及透射电子显微镜(TEM)分析了其粒径和形貌.采用荧光光谱仪与振动样品磁强计(VSM)测试证明该纳米粒子具备双模态成像功能.共聚焦成像(CLSM)的测试结果表明,该纳米试剂对动脉粥样硬化斑块中的巨噬细胞具有靶向性,MTT法的测试结果表明该探针分子具有良好的生物相容性.该双模态探针具有靶向性好、分辨率高及使用便捷的特点,是一种性能优异的双模态检测纳米试剂.     

扫描离子电导显微镜的原理及应用

扫描离子电导显微镜(SICM)是一种扫描探针显微技术,通过测定超微玻璃管探针的离子电流,它能够非接触地扫描样品表面,进而研究样品的形貌及性质。SICM具有成像分辨率高、探针易于制备和对被成像物体无损伤等特点,特别适用于研究生理条件下的活体细胞,是一种与扫描电化学显微镜及原子力显微镜互补的扫描探针显微镜技术。SICM能够对软界面及表面,如活细胞表面的显微结构,进行高分辨率成像;并能够与其它技术联用,研究细胞形貌与功能的关系;还能控制沉积特定分子,实现纳米尺度的显微操作与加工。本文对SICM的发展历史、仪器构造、基本原理及应用进行了综述。     

Ultra-sensitive detection of prion protein with a long range resonance energy transfer strategy

An ultra-sensitive detection strategy for prion protein is proposed based on the long range resonance energy transfer (LrRET) from quantum dots (QDs) to the surface of gold nanoparticles (AuNPs), in which process energy donor-acceptor separation distance ranges from 9 to 22 nm.     

一种基于纳米二氧化硅增强凝集反应的压电免疫传感器

本文提出了一种基于抗体包被纳米粒子的简单快速的压电免疫凝集法,用于蛋白质检测。该方法原理是利用羊抗人IgG（G-anti-hIgG）包被的二氧化硅（或金）纳米粒子和人IgG（hIgG）发生免疫凝集反应而使得压电晶体频率发生改变进行测定。当凝集反应发生时,修饰在探针表面的G-anti-hIgG通过hIgG与G-anti-hIgG包被的纳米粒子结合,将质量效应和粘弹性因素叠加作用于压电晶体。结果表明这使得背景值大幅减小而信号明显增强。另外,对修饰后了抗体及结合免疫复合物的探针表面进行了SEM表征,对使用聚乙二醇作为增敏剂和实验最佳离子强度、pH值进行了优化选择。该传感器检测hIgG线性范围是0.26-16.7 mg mL-1,最低检出限为84 ng mL-1。     

Utilizing NMR and EPR spectroscopy to probe the role of copper in prion diseases

Copper is an essential nutrient for the normal development of the brain and nervous system, although the hallmark of several neurological diseases is a change in copper concentrations in the brain and central nervous system. Prion protein (PrP) is a copper‐binding, cell‐surface glycoprotein that exists in two alternatively folded conformations: a normal isoform (PrP^C) and a disease‐associated isoform (PrP^Sc). Prion diseases are a group of lethal neurodegenerative disorders that develop as a result of conformational conversion of PrP^C into PrP^Sc. The pathogenic mechanism that triggers this conformational transformation with the subsequent development of prion diseases remains unclear. It has, however, been shown repeatedly that copper plays a significant functional role in the conformational conversion of prion proteins. In this review, we focus on current research that seeks to clarify the conformational changes associated with prion diseases and the role of copper in this mechanism, with emphasis on the latest applications of NMR and EPR spectroscopy to probe the interactions of copper with prion proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

Influence of particle size on the binding activity of proteins adsorbed onto gold nanoparticles

We used optical extinction spectroscopy to study the structure of proteins adsorbed onto gold nanoparticles of sizes 5-60 nm and their resulting biological binding activity. For these studies, proteins differing in size and shape, with well-characterized and specific interactions-rabbit immunoglobulin G (IgG), goat anti-rabbit IgG (anti-IgG), Staphylococcal protein A, streptavidin, and biotin-were used as model systems. Protein interaction with gold nanoparticles was probed by optical extinction measurements of localized surface plasmon resonance (LSPR) of the gold nanoparticles. Binding of the ligands in solution to protein molecules already immobilized on the surface of gold causes a small but detectable shift in the LSPR peak of the gold nanoparticles. This shift can be used to probe the binding activity of the adsorbed protein. Within the context of Mie theory calculations, the thickness of the adsorbed protein layer as well as its apparent refractive index is shown to depend on the size of the gold nanoparticle. The results suggest that proteins can adopt different orientations that depend on the size of the gold nanospheres. These different orientations, in turn, can result in different levels of biological activity. For example, we find that IgG adsorbed on spheres with diameter ≥20 nm does not bind to protein A. This study illustrates the principle that the size of nanoparticles can strongly influence the binding activity of adsorbed proteins. In addition to the importance of this in cases of direct exposure of proteins to nanoparticles, the results have implications for proteins adsorbed to materials with nanometer scale surface roughness.     

Preparation and a time-resolved fluoroimmunoassay application of new europium fluorescent nanoparticles.

New silica-based europium fluorescent nanoparticles having surface amino groups were prepared by a covalent binding-copolymerization technique. In the nanoparticles, the fluorescent Eu3+ chelate molecules were covalently bound to silicon atoms to protect the nanoparticles from dye leaking in bio-applications. The amino groups on the surface of nanoparticles made the surface modification and bioconjugation of nanoparticles easier. The nanoparticles were characterized and developed as a new type of fluorescence probe for a highly sensitive time-resolved fluoroimmunoassay (TR-FIA) of human hepatitis B surface antigen (HBsAg).     

Tunable inhibition and denaturation of alpha-chymotrypsin with amino acid-functionalized gold nanoparticles

Water-soluble gold nanoparticles bearing diverse l-amino acid terminals have been fabricated to probe the effect of receptor surface on protein surface binding. The interaction of these nanoparticles with alpha-chymotrypsin (ChT) was investigated by activity assay, gel electrophoresis, zeta-potential, circular dichroism, and fluorescence spectroscopy. The results show that both electrostatic and hydrophobic interactions between the hydrophobic patches of receptors and the protein contribute to the stability of the complex. The microscopic binding constants for these receptor-protein systems are 10(6)-10(7) M(-1), with the capacity of the nanoparticle receptors to bind proteins determined by both their surface area and their surface charge density. Furthermore, it is found that the hydrophilic side chains destabilize the ChT structure through either competitive hydrogen bonding or breakage of salt bridges, whereas denaturation was much slower with hydrophobic amino acid side chains. Significantly, correlation between the hydrophobicity index of amino acid side chains and the binding affinity and denaturation rates was observed.     

A novel route for immobilization of oligonucleotides onto modified silica nanoparticles

A novel approach for immobilization of probe oligonucleotides that uses zirconium phosphate modified silica nanoparticles is proposed. The surface modification of nanoparticles was carried out in two stages. Initially binding of Zr4+ to the surface of silica nanoparticles and later treated with phosphoric acid for terminal phosphate groups. Oligonucleotide probes modified with amine group at 5'-end were strongly binds to the phosphate terminated silica nanoparticles with imidazole in presence of 0.1 mol L(-1) EDC [N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide], as phosphate groups are more reactive towards amine group. Various studies, i.e., synthesis of silica nanoparticles, their surface modification, probe immobilization, measurement of hybridization and effect of bovine serum albumin (BSA) were carried out during optimization of reaction conditions. The significant reduction in the background signal was observed by treating the probe modified silica nanoparticles with bovine serum albumin prior to hybridization. The probe modified silica nanoparticles were retained their properties and the hybridization was induced by exposure of single-stranded DNA (ssDNA) containing silica nanoparticles to the complementary DNA in solution. The decrease in the fluorescence signal for one mismatch and three mismatch was observed upon hybridization of probe with target DNAs, while there was no response for the random target ssDNA under the same experimental conditions. The intensity of fluorescence signal was linear to the concentration of target DNA ranging from 3.9 x 10(-9) to 3.0 x 10(-6)mol L(-1). A detection limit of 1.22 x 10(-9) mol L(-1) of oligonucleotides can be estimated. The proposed hybridization assay is simple and possesses good analytical characteristics and it can provide an effective and efficient route in the development of DNA biosensors and biochips.     

Size-dependent carrier dynamics in CdS nanoparticles by femtosecond visible-pump/IR-probe measurements

Ultrafast photoexcited carrier dynamics in CdS nanoparticles prepared by an AOT/n-heptane reversed micelle system were investigated by a femtosecond visible-pump/mid-IR probe technique. A mid-IR probe beam was found to mainly probe the ultrafast dynamics of photoexcited electrons in the conduction band. Dispersions of CdS nanoparticles with 8 different mean diameters from 2.9 to 4.1 nm were prepared by tuning the mole ratio between water and AOT (W = [H(2)O]/[AOT]) in the reversed micelle systems. The excited state lifetime strongly depended on the mean size of CdS nanoparticles with a maximum around a mean diameter of 3.5 nm. This result was explained by considering the balance between the carrier recombination rates via surface states and those via interior states. The relationship between the excited state lifetime and the size of CdS nanoparticles was drastically changed when the surface was terminated by thiol molecules.     

Detection protein biomarker with gold nanoparticles functionalized hollow mesoporous Prussian blue nanoparticles as electrochemical probes

Gold nanoparticles functionalized hollow mesoporous Prussian blue nanoparticles(Au@HMPB NPs)were synthesized and its peroxidase-like activity was explored for electrochemical probe.The Au@HMPB NPs can reduce H2O2 low detection potential of-0.1 V with high sensitivity.After physically adsorption of antibodies onto the gold nanoparticle surface,the functionalized nanoparticles were turned into immuno-probe.The soluble a-chain of interleukin-2(IL-2)receptor(sCD25)was chosen as a model protein biomarker to test the performance of the probe.sCD25 in the samples were captured and enriched by capture anti-CD25 antibody functionalized magnetic nanospheres.Detection antibody functionalized Au@HMPB can then be linked onto the nanospheres and generate electrochemical current towards H2O2 reduction.The electrochemical responses to 1 mmol/L H2O2 was increased with the increasing concentration of CD25.     

金银合金纳米粒子表面处理及其表面增强拉曼光谱研究

采用水合肼还原的方法制备了金银比例为1∶1的金银合金纳米粒子, 紫外可见吸收光谱显示合成的溶胶只有一个介于金和银之间的吸收峰, 证明了合金结构的形成. 通过氨基耦联方法将合金纳米粒子组装到硅片表面, 利用氯金酸与合金中银的反应对基底上合金纳米粒子表面进行了改性处理. 以吡啶为探针分子, 研究了表面处理前后基底的SERS效应的差别, 结果表明随着浸泡时间延长, 信号强度先逐渐增强后降低至不变, 这与合金纳米粒子表面结构的变化有关, 氯金酸与表面银的反应经历了两个过程, 即粒子表面形成小的孔洞(去合金过程)和AgCl(s)在粒子表面的沉积, 前者有利于SERS效应的提高, 而后者导致SERS效应快速衰减.