Use of nanoparticles for studying the conformations of submembrane hemoglobin

The advantages and specific features of integrated application of atomic force microscopy, laser interference microscopy, and Raman microscopy in the study of erythrocytes are discussed. For successful application of Raman microscopy in the surface-enhanced mode, use was made of silver colloids. The dependence of the enhancement of Raman signals on silver nanoparticle size is demonstrated. The use of developed methods in clinical diagnostics is discussed.     

尿液表面增强拉曼光谱的初步研究

利用紫外与可见分光光度计测量银溶胶与尿液的吸收谱,采用拉曼光谱测量系统检测并研究分析了尿液加入银胶前后的拉曼光谱.基于表面增强技术,尿液的拉曼光谱信号得到显著增强,尿液中微弱的尿酸SERS信号被成功检测.文中对尿液的拉曼峰进行了谱峰归属,并分析了晨尿与夜尿的SERS谱.对晨尿的检测具有更高的可信度和信噪比.研究结果表明...     

Characterization and application of Raman labels for confocal Raman microspectroscopic detection of cellular proteins in single cells

A method using confocal Raman microspectroscopy for the detection of cellular proteins in single intact cells was developed. Two approaches were used to improve the detection of these cellular components. First, compounds with high Raman scattering were investigated for potential use as Raman labels. Raman labels were conjugated to either biomolecules or biotin and used as markers in the detection of cellular enzymes and receptors. Second, silver colloids were used to increase the surface-enhanced Raman scatter (SERS) of these Raman labels. Cresyl violet and dimethylaminoazobenzene are Raman labels that provide very sensitive SERS detection by a confocal Raman microscope with a HeNe laser at wavelength of 632.8 nm. The detection of 12-lipoxygenase and cyclooxygenase-1 in single bovine coronary artery endothelial cells and the binding of angiotensin II to its receptors in zona glomerulosa cells was demonstrated.     

银胶中三螺旋RNA poly（rU）．poly（rA）．poly（rU）的付立叶表面增强拉曼光谱研究

采用近红外付立叶拉曼光谱研究了三螺旋ＲＮＡ（ｒＵ）．ｐｏｌｙ（ｒＡ）．ｐｏｌｙ（ｒＵ）在溶液中的构象和在银胶中的表面增强拉曼散行为。结果表明在溶液中,该三螺旋ＲＮＡ分子中以Ｗａｔｓｏｎ－Ｃｒｉｃｋ碱基酸对的两条链处于Ａ－构型,而第二条嘧啶链处于Ｃ２’－ｅｎｄｏ／ａｎｔｉ构象。在银胶中,该三螺旋ＲＮＡ的表面增强拦曼效应明显。与溶液状态下相比,８３５和８１９ｃｍ＾－１谱带的出现暗示该三螺旋ＲＮＡ吸附到     

Fluorescence enhancement of fluorophores tethered to different sized silver colloids deposited on glass substrate

We studied fluorescence enhancements of fluorescein tethered to silver colloids of different size. Thiolated 23-mer oligonucleotide (ss DNA-SH) was bound selectively to silver colloids deposited on 3-aminopropyltriethoxysilane (APS)-treated quartz slides. Fluorescein-labeled complementary oligonucleotide (ss Fl-DNA) was added in an amount significantly lower than the amount of unlabeled DNA tethered to the colloids. The hybridization kinetics, observed as an increase in fluorescence emission, on small (30-40 nm) and large (> 120 nm) colloids were similar. However, the final fluorescence intensity of the sample with large colloids was about 50% higher than that observed for the sample with small colloids. The reference sample without ss DNA-SH was used to estimate the fluorescence enhancements of fluorescein tethered to the small colloids (E = 2.7) and to the large colloids (E = 4.1) due to its steady fluorescence signal. The proposed method, based on controlled hybridization with minimal amount of fluorophore labeled ss DNA, can be used to reliably estimate the fluorescence enhancements on any silver nanostructures.     

Surface enhanced Raman scattering of a lipid Langmuir monolayer at the air-water interface

Surface enhanced Raman spectra were recorded from a phospholipid monolayer directly at the air-water interface. We used an organized monolayer of negatively charged tetramyristoyl cardiolipins as a template for the electrochemical generation of silver deposits. This two-dimensional electrodeposition of silver under potentiostatic control was the substrate for enhancement of Raman spectra. We report the optimized conditions for the Raman enhancement, the microscopic observations of the deposits, and their characterization by atomic force microscopy. Laser excitation at 514.5 nm leads to intense and reproducible surface enhanced Raman scattering spectra recorded in situ from one monolayer of cardiolipin, using 0.5 mol % of 10N nonyl acridine orange or 5 mol % of acridine in the film, and demonstrates the possibility of estimating the pH at the metal/phospholipidic film interface.     

Modifying and Fine Controlling of Silver Nanoparticle Nucleation Sites and SERS Performance by Double Silicon Etching Process

Different forms of modified and well-controlled plasmonic silver nanoparticles (AgNPs) were synthesized by silver ion reduction process of porous silicon (PS). Fine control of PS surface morphology was accomplished by employing two etching processes: light-induced etching (LIE) and photo electrochemical etching (PECE). The idea was to prepare excellent and reproducible surface-enhanced Raman scattering (SERS) substrates with high enhancement performance. PS surface modification was employed to create efficient and nearly uniformly distributed AgNP hotspot regions with very high specific surface areas. Reproducibility deviation of no more than 5% and enhancement factor of 1.2 × 10¹⁴ were obtained by SERS measurements at very low, rhodamine 6G (R6G) dye, concentration 10^?15 M. The PS morphology SERS substrate was well discussed and analyzed using field emission scanning electron microscopy (FE-SEM), X-ray diffraction spectroscopy (XRD), and Raman measurements.     

One- and two-photon induced fluorescence of Pacific Blue-labeled human serum albumin deposited on different core size silver colloids

We studied one- and two-photon induced fluorescence of Pacific Blue (PB)-labeled human serum albumin (HSA) in the presence of different size silver colloids. The PB fluorescence emission intensity was observed with small (30-40 nm) and large (about 120 nm) colloids and compared with PB emission in absence of colloids. For the system with a small core size colloids we did not detect any fluorescence enhancement with one-photon excitation and the enhancement observed with two-photon excitation was about 2.5-fold. In contrast, for large silver colloids we observed about a 2-fold increase in PB fluorescence brightness for one-photon excitation, and the enhancement with two-photon excitation excided 13-folds. Much stronger increases in brightness observed with two-photon excitation, compared to one-photon excitation, indicate a dominant role of enhanced local field in fluorescence enhancement on silver colloids in solutions.     

An intracellular silver deposition method for targeted detection and chemical analysis of uncultured microorganisms

The vast majority of environmental bacteria remain uncultured, despite two centuries of effort in cultivating microorganisms. Our knowledge of their physiology and metabolic activity depends to a large extent on methods capable of analyzing single cells. Bacterial identification is a key step required by all currently used single-cell imaging techniques and is typically performed by means of fluorescent labeling. However, fluorescent cells cannot be visualized by ion- and electron microscopy and thus only correlative, indirect, cell identification is possible. Here we present a new method of bacterial identification by in situ hybridization coupled to the deposition of elemental silver nanoparticles (silver-DISH). We show that hybridized cells containing silver can be directly visualized by light microscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy, secondary ion mass spectrometry (nanoSIMS), and confocal Raman micro-spectroscopy. Silver-DISH did not alter the isotopic (¹³C) and elemental composition of stable-isotope probed cells more than other available hybridization methods, making silver-DISH suitable for broad applications in stable-isotope labeling studies. Additionally, we demonstrate that silver-DISH can induce a surface-enhanced Raman scattering (SERS) effect, amplifying the Raman signal of biomolecules inside bacterial cells. This makes silver-DISH the only currently available method that is capable of delivering a SERS-active substrate inside specifically targeted microbial cells.     

Intracellular dynamics of topoisomerase I inhibitor,CPT-11, by slit-scanning confocal Raman microscopy

Most molecular imaging technologies require exogenous probes and may have some influence on the intracellular dynamics of target molecules. In contrast, Raman scattering light measurement can identify biomolecules in their innate state without application of staining methods. Our aim was to analyze intracellular dynamics of topoisomerase I inhibitor, CPT-11, by using slit-scanning confocal Raman microscopy, which can take Raman images with high temporal and spatial resolution. We could acquire images of the intracellular distribution of CPT-11 and its metabolite SN-38 within several minutes without use of any exogenous tags. Change of subcellular drug localization after treatment could be assessed by Raman imaging. We also showed intracellular conversion from CPT-11 to SN-38 using Raman spectra. The study shows the feasibility of using slit-scanning confocal Raman microscopy for the non-labeling evaluation of the intracellular dynamics of CPT-11 with high temporal and spatial resolution. We conclude that Raman spectromicroscopic imaging is useful for pharmacokinetic studies of anticancer drugs in living cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Direct spectroscopic determination of functional sulphydryl groups on intact cell surfaces by surface-enhanced resonance Raman scattering

Semi-quantitative and direct determination of labelled sulphydryl groups on the surface of intact erythrocytes has been accomplished for the first time with surface-enhanced resonance Raman scattering (SERRS). The method, which involves the use of citrate-reduced silver colloids, is sensitive and selective. A 10^–8 M effective concentration of picomole quantities of sulphydryl groups was determined in the presence of the normally overwhelming signal from haemoglobin. This seminal study suggests that SERRS may be applied to other in situ, site-directed labelling experiments. Correspondence to: W.E. Smith     

Antibody-based SERS diagnostics of fhit protein without label

Surface-enhanced Raman scattering (SERS) is a particularly promising technique that has the potential to perform highly selective and sensitive in situ measurements of antibody-antigen reactions. This work describes the use of silver (Ag) colloids for immunoassay-based SERS detection of the fragile histidine triad (Fhit) protein. Alterations in Fhit protein expression have been associated with several human cancers, and, thus, the detection of Fhit protein is important because it can potentially be used as a cancer diagnostic biomarker, for both cancer detection and therapy.     

Comparison and Evaluation of Silver Probe Preparation Techniques for Tip-Enhanced Raman Spectroscopy

In this work, the different procedures for the fabrication of Ag probes for tip-enhanced Raman spectroscopy (TERS) in a top illumination/detection setup are proposed and tested. We focus on technologically simple methods allowing Si tips coated with plasmonic silver nanostructures and bulk metal Ag tips with good shape reproducibility to be produced for atomic force microscopy (AFM) feedback setup. The preparation of Ag TERS probes was based on chemical deposition and vacuum sputtering of Ag on the tips of commercially available Si cantilevers. A straightforward technique for the fabrication of bulk metal Ag probes by the electrochemical etching of Ag microwires was also proposed. Chemically coated, sputtered, and electrochemically etched TERS tips were characterized by scanning electron microscopy (SEM). The produced tips were tested for TERS measurements using graphene oxide (GO) as the target analyte in a top illumination setup. A comparative analysis of enhancement factors (EF) for the different types of tips (probes) is presented in this work.     

Raman spectroscopy of model membrane monolayers of dipalmitoylphosphatidic acid at the air-water interface using surface enhancement from buoyant thin silver films

A novel method for the acquisition of surface enhanced Raman (SER) spectra of model membranes of dipalmitoylphosphatidic acid (DPPA) in Langmuir layers at the air-water interface is reported. The approach is based on the electrochemical formation of a buoyant thin layer of coalesced silver colloids in the vicinity of the phosphatidic acid head groups at the interface. This Ag layer is an excellent platform for SER scattering, which shows the spectral features from all parts of the molecule and water between the Ag surface and the DPPA layer. The observation of the spectral response from the phosphatidic acid head groups is of particular significance, allowing insight into their chemical state and orientation at the air-water interface.     

Methods of detection and identification of manufactured nanoparticles

The actual methods of detection and identification of manufactured nanoparticles in both simple and complex multi-component matrix for assessing biological effects and safety of nanotechnology products have been reviewed. The detection of priority species of biologically active nanoparticles, which include fullerenes, singleand multi-walled carbon nanotubes, nanoparticles of silver, gold, titanium oxide, aluminum, cerium, zinc and silicon, has been given a special attention. The requirements for sample preparation have been discussed. The results of the successful application for the detection of manufactured nanoparticles in biosamples with methods of scanning and transmission electron microscopy, confocal laser scanning microscopy, atomic force microscopy, scanning tunneling microscopy, size exclusion chromatography, field-flow fractionation, electrophoretic, light scattering, spectrophotometry, fluorescent spectroscopy, X-ray and other spectrometry, mass spectrometry, “particle counters”, immunochemistry have been reviewed. The possibilities and limitations of different techniques, and their complementarity have been analyzed.     

Surface-enhanced Raman and steady fluorescence study of interaction between antitumoral drug 9-aminoacridine and trypsin-like protease related to metastasis processes, guanidinobenzoatase

Fluorescence spectroscopy and surface-enhanced Raman spectroscopy (SERS) were applied to study the interaction of the antitumoral drug 9-aminoacridine (9AA) with a trypsin-like protease guanidinobenzoatase (GB) extracted from a mouse Erlich tumor. As a consequence of this interaction, a strong 9AA exciplex emission was detected in the emission fluorescence spectra at certain drug and enzyme concentrations. A SERS study was accomplished on silver colloids at several excitation wavelengths in order to obtain more information about the interaction mechanism. The results derived from Raman spectroscopy indicated that 9AA in the amino monomeric form may interact with the enzyme by means of two different bonds: an ionic bond with a negatively charged amino acid and a ring stacking interaction with an aromatic residue placed in the catalytic site of GB. This interaction mechanism was responsible for a strong exciplex emission detected at a longer wavelength than the expected value of the normal fluorescence emission. Moreover, the GB concentration dependence of the interaction suggested that the drug was sensitive to the quaternary structure of the enzyme.     

Theoretical Study of the Local Surface Plasmon Resonance Properties of Silver Nanosphere Clusters

The local surface plasmon resonance properties in systems consisting of silver nanosphere clusters are studied by Green’s function. The extinction, absorption, and scattering efficiencies band of two, three, and more silver nanospheres clusters are discussed in detail. The clusters show new types of the local surface plasmon resonances compared with single silver nanosphere. Our results suggest that the resonances depend strongly on individual particles’ characteristics such as their shapes, gap distances, directions and polarizations of incident light waves, and the number of clusters. The spectrum shows that equilateral triangle nanospheres has a good absorption peak, while the better red-shifted with three aligned nanospheres. In addition, the distributions of electric field intensity for three and four touched silver nanospheres are also investigated. The study is useful to broaden the application scope of Raman spectroscopy and nanooptics.     

Immunogold silver staining for light microscopy

 The immunogold silver staining method (IGSS) is widely used as a sensitive and specific immunohistochemical visualisation technique. IGSS involves the specific deposition of metallic silver at the site of immunogold labelling and provides a means of visualisation at low magnification by light or electron microscopy. Silver developers for IGSS rapidly deposit metallic silver only at the site of heavy metals, including gold and silver, because of their catalytic activity. The developing solution contains the silver ions and reducing agent necessary for this reaction. Using different silver salts as ion donors and by selecting an appropriate temperature and pH, visible amounts of silver can be deposited in a few minutes at the site of colloidal gold labelling while little non-specific background deposition occurs. Inclusion of protective colloids in the solution can also be used to control the reaction. Although studies of the chemical basis of silver deposition around unlabelled colloidal gold date back to 1939, immunogold enhancement by silver was established in 1983. The IGSS method evolved from the combination of disparate photographic, histochemical and immunogold techniques which have been effectively combined and optimised over the last 10 years to provide a visualisation system which is well suited to many immunohistochemical studies. Accepted: 29 April 1996     

Rapid detection of food- and waterborne bacteria using surface-enhanced Raman spectroscopy coupled with silver nanosubstrates

Development of rapid and sensitive methods to detect pathogens is important to food and water safety. This study aimed to detect and discriminate important food- and waterborne bacteria (i.e., Escherichia coli O157:H7, Staphylococcus epidermidis, Listeria monocytogenes, and Enterococcus faecelis) by surface-enhanced Raman spectroscopy (SERS) coupled with intracellular nanosilver as SERS substrates. An in vivo molecular probing using intracellular nanosilver for the preparation of bacterial samples was established and assessed. Satisfactory SERS performance and characteristic SERS spectra were obtained from different bacterial samples. Distinctive differences were observed in SERS spectral data, specifically in the Raman shift region of 500–1,800 cm⁻¹, and between bacterial samples at the species and strain levels. The detection limit of SERS coupled with in vivo molecular probing using silver nanosubstrates could reach the level of single cells. Experiments with a mixture of E. coli O157:H7 and S. epidermidis for SERS measurement demonstrate that SERS could be used for classification of mixed bacterial samples. Transmission electron microscopy was used to characterize changes of morphology and cellular composition of bacterial cells after treatment of intracellular nanosilver. The results indicate that SERS coupled with intracellular silver nanosubstrates is a promising method for detection and characterization of food- and waterborne pathogenic and non-pathogenic bacterial samples.     

微生物合成纳米银的一般方法及产物性质鉴定与生产应用

纳米银具有独特的理化性质,在化学、医药等领域应用广泛,但使用化学和物理法生产纳米银毒性较强且污染严重,因此,微生物法成为了一种可供替代的绿色生产技术。近年来,微生物法合成纳米银的报道逐渐增多,对其反应条件和产物性质的研究趋于成熟,纳米银也开始与多个应用领域相结合。归纳现有微生物合成方法的规律,阐述产物的性质及应用,比较其与传统材料的优势与不足,将有助于推动微生物与纳米技术的结合与进步。基于国内外学者的报道和本课题组所开展的相关研究,本文对微生物法合成纳米银的一般检验手段和功能鉴定方法进行综述,并就纳米银的应用展开设想与讨论,以期为微生物法合成纳米银的深入研究和优化改进提供参考。