Ultra-sensitive colorimetric method to quantitate hundreds of polynucleotide molecules by gold nanoparticles with silver enhancement.

An ultra-sensitive colorimetric method to quantitate hundreds of polynucleotide molecules by gold nanoparticles with silver enhancement has been developed. The hybridization products from the target polynucleotides with biotin-labeled probes and gold nanoparticle-functioned oligonucleotides were immobilized to microplates via avidin-biotin system, and the absorbance signals of gold nanoparticles were amplified by silver enhance solution. This sandwich colorimetric assay can detect as few as 600 molecules for single-strand oligonucleotides and as few as 6000 molecules for double-strand polynucleotides in a 50 microL reaction system.     

Protein Enhanced Near-Infrared Fluorescence of AuNPs and Its Application for Protein Determination

The 15～25 nm water soluble and stable gold nanoparticles were synthesized and studied for their spectral properties and interactions with proteins. Results showed that 15 nm gold nanoparticles can emit near infrared fluorescence with an emission peak of 811.2 nm under the excitation of 538 nm. The study also showed that proteins can obviously enhance the near infrared fluorescence intensity of gold nanoparticles. Under the optimized conditions, there is a linear relationship between the fluorescence intensity enhancement of the system and the concentrations of the proteins, which can be used in a new method for the determination of trace proteins. The mechanisms of the interaction and the fluorescence enhancement of the nano-gold-protein system were also studied.     

Sensing lipid bilayer formation and expansion with a microfabricated cantilever array

We show that cantilever array sensors can sense the formation of supported phospholipid bilayers on their surface and that they can monitor changes in mechanical properties of lipid bilayers. Supported lipid bilayers were formed on top of microfabricated cantilevers by vesicle fusion. The formation of bilayers led to a bending of the cantilevers of 70-590 nm comparable to a surface stress of 27-224 mN/m. Physisorption of bilayers of DOPC and other bilayers on the silicon oxide surface of cantilevers led to a tensile bending of about 70 nm whereas formation of chemisorbed bilayers of mixed thiolated (DPPTE) and non-thiolated lipids (DOPC) on the gold side of cantilevers led to a compressive bending of nearly 600 nm which depended on the ratio of DPPTE to DOPC. First results on bending of bilayer-covered cantilevers due to their interaction with the pore-forming peptide melittin are shown. The results demonstrate that cantilever sensors with immobilized bilayers can be used as model systems to investigate mechanical properties of cellular membranes and may be used for screening of membrane processes involving modification, lateral expansion, or contraction of membranes.     

Oriented attachment and membrane reconstitution of His-tagged cytochrome c oxidase to a gold electrode: in situ monitoring by surface-enhanced infrared absorption spectroscopy

A novel concept is introduced for the oriented incorporation of membrane proteins into solid supported lipid bilayers. Recombinant cytochrome c oxidase solubilized in detergent was immobilized on a chemically modified gold surface via the affinity of its histidine-tag to a nickel-chelating nitrilo-triacetic acid (NTA) surface. The oriented protein monolayer was reconstituted into the lipid environment by detergent substitution. The individual steps of the surface modification, including (1) chemical modification of the gold support, (2) adsorption of the protein, and (3) reconstitution of the lipid bilayer, were followed in situ by means of surface-enhanced infrared absorption spectroscopy (SEIRAS) and accompanied by normal-mode analysis. The high surface sensitivity of SEIRAS allows for the identification of each chemical reaction process within the monolayer at the molecular level. Finally, full functionality of the surface-tethered cytochrome c oxidase was demonstrated by cyclic voltammetry after binding of the natural electron donor cytochrome c.     

Electrochemical and microscopic characteristics of thiolipid layers as simple models of cell membranes

The thiolipid 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) has been proposed as a component of a simple model of cell membranes, which can be used for the studies of the interactions with drugs and other biologically important species. Depending on the deposition technique, Langmuir-Blodgett method or self-assembly, the obtained model membranes exhibit differences in the organization and properties, as shown by electrochemical techniques. The surface concentration and area per molecule of DPPTE model membranes were determined using chronocoulometry, which gives more reliable results than the widely used reductive desorption method. The mean surface concentration of self-assembled DPPTE monolayer deposited on gold electrode is equal to 4.52 × 10(-10) mol·cm(-2), which corresponds to the area per molecule of 36.7 ?(2). Moreover, model membranes prepared by means of LB method tend to be less compact than self-assembled DPPTE monolayers. Adsorption/desorption behavior of the DPPTE molecules on Au(111) was also visualized by EC-STM method. At the beginning of the process at negative potentials, the physisorbed molecules formed a flat-lying adlayer. Changing the potential in the positive direction resulted in the formation of Au-S bonds, and as a consequence the upstanding phase with higher packing density was observed. The thickness of such a layer determined by atomic force microscopy method is equal to 2.08 nm and corresponds to that of a monomolecular film.     

Adsorption characteristics of thionine on gold nanoparticles

Adsorption characteristics of thionine on gold nanoparticles have been studied by using UV-vis absorption spectroscopy, fluorescence spectroscopy, transmission electron microscopy (TEM), cyclic voltammetry and Fourier transform infrared spectroscopy. With the increasing concentration of gold nanoparticles, the absorption peak intensity of H-type dimers of thionine increases continuously, whereas that of monomers of thionine first increases and then decreases. The addition of gold nanoparticles makes the equilibrium between the monomer and H-type dimer forms of thionine move toward the dimer forms. Furthermore, the adsorption behavior of thionine on gold nanoparticles is also influenced by temperature. TEM images show that the addition of thionine results in an obvious aggregation, and further support the absorption spectral results. The fluorescence intensity of adsorbed thionine is quenched by gold nanoparticles due to the electronic interaction between thionine molecules and gold nanoparticles. Cyclic voltammetric and infrared spectroscopic studies show that the nitrogen atoms of both of the NH2 moieties of thionine strongly bind to the gold nanoparticle surfaces through the electrostatic interaction of thionine with gold nanoparticles. For 15-20 nm particles, the number of adsorbed thionine molecules per gold nanoparticle is about 7.66 x 10(4). Thionine molecules can not only bind to a particle to form a compact monolayer via both of the NH2 moieties, but they can also bind to two particles via their two NH(2) moieties, respectively.     

Hemoglobin on phosphonic acid terminated self-assembled monolayers at a gold electrode: immobilization, direct electrochemistry, and electrocatalysis

Phosphonic acid (--PO(3)H(2)) terminated self-assembled monolayers (SAMs) on a gold surface were used as a functional interface to immobilize hemoglobin (Hb). In situ surface-enhanced infrared absorption spectroscopy (SEIRAS) measurements show that Hb immobilization is a sluggish process due to formation of multilayer Hb structures on the PO(3)H(2)-terminated SAMs, as revealed by ellipsometry, atomic force microscopy (AFM), and cyclic voltammetry (CV). In the multilayered Hb film, the innermost Hb molecules can directly exchange electrons with the electrode, whereas Hb beyond this layer communicates electronically with the electrode via protein-protein electron exchange. In addition, electrochemical measurements indicate that immobilization of Hb on the PO(3)H(2)-terminated SAMs is not driven by the electrostatic interaction, but likely by hydrogen-bonding interaction. The immobilized Hb molecules show excellent bioelectrocatalytic activity towards hydrogen peroxide, that is, the PO(3)H(2)-terminated SAMs are promising for construction of third-generation biosensors.     

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.     

Application of thiolated gold nanoparticles for the enhancement of glucose oxidase activity

Glucose oxidase (GOx) has been covalently immobilized onto chemically synthesized thiolated gold nanoparticles (5-8 nm) via N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The lower value of the Michaelis-Menton constant obtained for the immobilized (3.74 mM) GOx compared with that for the free (5.85 mM) GOx suggests significant enhancement in the activity of GOx attached to thiolated gold nanoparticles. The covalently immobilized GOx thiolated nanoparticles exhibit a response time of 30 s, a shelf life of more than 6 months, and improved tolerance to both pH and temperature.     

气液界面磷脂单分子膜的表面增强拉曼光谱

采用纳米银胶作为成膜亚相, 原位获得了十八胺单分子膜、十八胺/卵磷脂复合膜的表面增强拉曼信号.研究表明,增强主要来源于亚相中的银粒子与成膜分子之间较强的作用.通过在磷脂膜内添加十八胺分子辅助增强而获取了卵磷脂的分子振动信息.     

Detection of Vibrational Spectroscopic Biomarkers of the Effect of Gold Nanoparticles on Wheat Seedlings Using Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy

The aim of this study is to evaluate the biochemical changes in the leaves of wheat seedlings exposed to gold nanoparticles (AuNPs) nondestructively and rapidly using attenuated total reflectance Fourier transform infrared spectroscopy and laser-induced fluorescence. The 18?nm size gold nanoparticles are synthesized by citrate reduction. For analyzing the effect of gold nanoparticles on wheat seedlings, the treatment of gold nanoparticles was applied to the seedlings through roots and following the spectroscopic measurement of biochemical signatures. The laser-induced fluorescence measurement has been performed to access the effect of gold nanoparticles on the chlorophyll concentration of wheat seedlings. The decrease in the fluorescence intensity and the fluorescence intensity ratio on the treatment of gold nanoparticles indicates increase in the concentration of chlorophyll in the leaves of wheat seedlings. The attenuated total reflectance Fourier transform infrarred spectroscopy in combination with principal component analysis has been used to visualize the biochemical changes in the cellulose, hemicellulose, pectin, lignin, amino acids, proteins, and lipid of the leaves of wheat seedlings by recording infrared spectra in the region from 4000 to 400?cm^?1. Principal component analysis applied to the preprocessed infrared data clearly distinguishes the spectral variability between control and gold nanoparticle treated seedlings. The study shows that exposure of gold nanoparticles increases the concentrations of cellulose, hemicelluloses, pectin, and lignin in the leaves of wheat seedlings. The increase in these chemicals indicates the modulation of cell walls of the wheat seedlings by the gold nanoparticle treatment. The exposure to gold nanoparticles also enhances the expression of lipid and proteins in the leaves of wheat seedlings.     

Detection of polycyclic aromatic hydrocarbon (PAH) compounds in artificial sea-water using surface-enhanced Raman scattering (SERS)

This paper reports an accurate synthesis of surface-enhanced Raman scattering (SERS) active substrates, based on gold colloidal monolayer, suitable for in situ environmental analysis. Quartz substrates were functionalized by silanization with (3-mercaptopropyl)trimethoxysilane (MPMS) or (3-aminopropyl)trimethoxysilane (APTMS) and they subsequently reacted with colloidal suspension of gold metal nanoparticles: respectively, the functional groups SH and NH₂ bound gold nanoparticles. Gold nanoparticles were prepared by the chemical reduction of HAuCl₄ using sodium tricitrate and immobilized onto silanized quartz substrates. Active substrate surface morphology was characterized with scanning electron microscopy (SEM) measurements and gold nanoparticles presented a diameter in the range 40-100 nm. Colloidal hydrophobic films, allowing nonpolar molecule pre-concentration, were obtained. The surfaces exhibit strong enhancement of Raman scattering from molecules adsorbed on the films. Spectra were recorded for two PAHs, naphthalene and pyrene, in artificial sea-water (ASW) with limits of detection (LODs) of 10 ppb for both on MPMS silanized substrates.     

Near‐Infrared Light Triggered‐Release in Deep Brain Regions Using Ultra‐photosensitive Nanovesicles

Remote and minimally‐invasive modulation of biological systems with light has transformed modern biology and neuroscience. However, light absorption and scattering significantly prevents penetration to deep brain regions. Herein, we describe the use of gold‐coated mechanoresponsive nanovesicles, which consist of liposomes made from the artificial phospholipid Rad‐PC‐Rad as a tool for the delivery of bioactive molecules into brain tissue. Near‐infrared picosecond laser pulses activated the gold‐coating on the surface of nanovesicles, creating nanomechanical stress and leading to near‐complete vesicle cargo release in sub‐seconds. Compared to natural phospholipid liposomes, the photo‐release was possible at 40 times lower laser energy. This high photosensitivity enables photorelease of molecules down to a depth of 4 mm in mouse brain. This promising tool provides a versatile platform to optically release functional molecules to modulate brain circuits.     

纳米金三明治结构调制细胞色素c电子传递特性的分子机理研究

以细胞色素c(Cyt c)为模型蛋白,采用表面增强红外吸收光谱监测了三明治结构所吸附的纳米金对氧化还原诱导的Cyt c表面增强红外差谱的改变.研究表明,在单层Cyt c分子表面组装纳米金,使得血红素的红外差谱特征峰明显增强,这归因于纳米金和血红素之间的电子传递.纳米金与Cyt c氧化还原活性中心血红素的相互作用加速了蛋白质的电子传递.这为实现并优化表面吸附蛋白质的直接电化学提供了一种新技术.     

不同受限状态下链硫醇的非线性光学响应

以和频(SFG)振动光谱技术探测了正十二硫醇(DDT)在不同受限状态下的分子振动信号,包括金属基底上的自组装单层(SAM)分子,放置在二氧化硅基底上的表面DDT化的金纳米粒子以及金纳米粒子的甲苯溶液.在三种状态下都探测到了来自于DDT分子的振动光谱,振动光谱的区别提供了在不同受限态下DDT分子的结构信息.在金属基底上DDT分子排列规整,放置在二氧化硅基底上的金纳米粒子表面的DDT分子具有一定的柔性,在空气-甲苯溶液界面金纳米粒子表面的DDT分子高度无序.此外,光谱实验显示,金纳米粒子表面的分子振动信号产生了局域场增强的效应,相对于金基底上的自组装单层分子而言,增强系数为102-103,取决于光谱的偏振组合.     

Revealing the kinetics of ionophore facilitating ion transport across lipid bilayers by surface enhanced infrared absorption spectroscopy

Ionophore can prominently improve the ion permeability of cell membrane and disrupt cellular ion homeostasis.Most studies regarding ionophore facilitating ion transmembrane transport focus on artificial liquid-liquid interfaces,which have large difference from the actual environment of cell membrane.Here,we construct a supported lipid bilayeron a gold nanoparticles film modified ZnSe prism as an appropriate model of cell membrane to investigate the dynamic of the ion transport facilitated by ionophore using surface enhanced infrared absorption spectroscopy(SEIRAS).We find that the ion transmembrane transport consists of two steps:The ion transmembrane transport starts with the association/disassociation between ion and ionophore at the edge of lipid bilayer;The second step is the transfer of ion-ionophore complex across lipid bilayer.Our results show that the complex transfer across the lipid bilayer is the rate determining step.     

Au纳米粒子表面附近偶氮苯分子的吸收光谱

我们将时空间含时密度泛函理论与经典的电动力学时空间有限差分技术相结合,对吸附在Au纳米粒子表面附近的偶氮苯分子顺式和反式两种同分异构体的光学性质进行了研究。计算结果显示,一方面表面等离子激元共振会增大其周围激光外场的强度,从而使得偶氮苯分子的吸收光谱得到增强,另一方面,吸附分子与金属纳米粒子之间的相互耦合作用也会对一些低能特殊谱峰的强度及位置产生影响,从而使得谱线的形状与单独的偶氮苯分子或Au纳米粒子并不相同。此外,等离子激元的增强效应会随着分子与金属纳米粒子之间的距离以及纳米粒子尺寸的变化而发生变化。     

Intracellular SERS hybrid probes using BSA–reporter conjugates

Surface-enhanced Raman scattering (SERS) hybrid probes are characterized by the typical spectrum of a reporter molecule. In addition, they deliver information from their biological environment. Here, we report SERS hybrid probes generated by conjugating different reporter molecules to bovine serum albumin (BSA) and using gold nanoparticles as plasmonic core. Advantages of the BSA-conjugate hybrid nanoprobes over other SERS nanoprobes are a high biocompatibility, stabilization of the gold nanoparticles in the biological environment, stable reporter signals, and easy preparation. The coupling efficiencies of the BSA–reporter conjugates were determined by MALDI-TOF-MS. The conjugates’ characteristic SERS spectra differ from the spectra of unbound reporter molecules. This is a consequence of the covalent coupling, which leads to altered SERS enhancement and changes in the chemical structures of the reporter and of BSA. The application of the BSA–reporter conjugate hybrid probes in 3T3 cells, including duplex imaging, is demonstrated. Hierarchical cluster analysis and principal components analysis were applied for multivariate imaging using the SERS signatures of the incorporated SERS hybrid nanoprobes along with the spectral information from biomolecules in endosomal structures of cells. The results suggest more successful applications of the SERS hybrid probes in cellular imaging and other unordered high-density bioanalytical sensing.

不同受限状态下链硫醇的非线性光学响应

以和频(SFG)振动光谱技术探测了正十二硫醇(DDT)在不同受限状态下的分子振动信号, 包括金属基底上的自组装单层(SAM)分子, 放置在二氧化硅基底上的表面DDT化的金纳米粒子以及金纳米粒子的甲苯溶液. 在三种状态下都探测到了来自于DDT分子的振动光谱, 振动光谱的区别提供了在不同受限态下DDT分子的结构信息. 在金属基底上DDT分子排列规整, 放置在二氧化硅基底上的金纳米粒子表面的DDT分子具有一定的柔性, 在空气-甲苯溶液界面金纳米粒子表面的DDT分子高度无序. 此外, 光谱实验显示, 金纳米粒子表面的分子振动信号产生了局域场增强的效应, 相对于金基底上的自组装单层分子而言, 增强系数为10²-10³, 取决于光谱的偏振组合.     

In situ monitoring of the orientated assembly of strep-tagged membrane proteins on the gold surface by surface enhanced infrared absorption spectroscopy

Surface enhanced infrared absorption spectroscopy (SEIRAS) has been employed to monitor the orientated assembly of a strep-tagged membrane protein on the gold surface via a streptavidin/biotin interlayer. The high surface sensitivity of SEIRAS allows for tracking the individual assembling steps on the molecular level. The sequence of surface modification steps comprises: (i) cross-linking of biotin to the self-assembled monolayer of cysteamine along the gold surface; (ii) adsorption of streptavidin to and desorption from the biotin layer; and (iii) adsorption of the strep-tagged membrane protein ecgltP (glutamate transporter of E. coli) on the streptavidin/biotin layer. The analysis of the SEIRA spectra reveals that the biotin layer undergoes a phase transition from an isotropic orientation to a densely packed layer during coupling to the cysteamine monolayer. Formation of the densely packed layer weakens the interaction between streptavidin and the biotin layer but yields a binding specificity of 80%. The specificity of strep-tagged ecgltP to the streptavidin layer is with 60% only modest. Nevertheless, the streptavidin/biotin interlayer reveals a higher regeneration propensity than the His-tag/Ni-NTA interlayer.