原子力显微镜-荧光显微镜联用技术在活细胞单分子检测中的应用

原子力显微镜(Atomic force microscopy,AFM)及荧光显微镜(Fluorescence microscopy,FM)是目前活细胞单分子分析检测中最常用的两种工具.结合两种显微镜的优势,发展高时空分辨、多功能的AFM-FM联用技术成为近年该领域的研究热点.本文简述了AFM单分子力谱和FM单分子荧光成像的原理,总结了AFM-FM联用系统在仪器研制方面的发展概况,并结合本课题组在应用AFM-FM联用技术研究细胞膜上配受体相互作用等方面的工作,介绍了其在活细胞单分子检测中的应用进展.     

Interfacial re-arrangement in initial microbial adhesion to surfaces

Upon initial microbial adhesion to a surface, multiple events occur that include interfacial re-arrangements in the region between an adhering organism and a surface. Application of physico-chemical mechanisms to explain microbial adhesion to surfaces requires better knowledge of the interfacial re-arrangement occurring immediately after adhesion than hitherto available.     

原子力显微镜在多糖结构研究中的进展

简述了原子力显微镜(AFM)的工作原理和特点,以及在多糖,特别是在淀粉结构研究中的进展。     

生物大分子的单分子力谱:在水溶液与非极性溶剂中的对照研究

生物大分子是构成生命的物质基础,在其天然环境——水溶液中一般以精确的超分子结构存在.迄今,人们已经合成了种类繁多的水溶性高分子.然而,鲜有合成高分子能够在水溶液中完成精确的超分子组装.与合成高分子相比,生物大分子是特殊而神奇的.为了研究生物大分子与水的相互作用,近年来作者以单分子力谱为主要的实验方法,开展了生物大分子在水溶液与非极性溶剂中的对照研究.研究表明,在非极性溶剂中,生物大分子的超分子结构失稳,转变为无超分子结构的状态.水是一个重要的开关,调控着生物大分子的超分子结构和功能.作者据此提出了生物大分子的水环境适应性概念和早期化学进化过程中水环境筛选生物大分子的假说,并认为水环境适应性是生物大分子和合成水溶性高分子的分水岭.对水和生物大分子的深入研究,将有望破解生命的更多奥秘.     

单分子水平高分子相互作用的AFM成像与单分子力谱研究

结合作者近期的研究工作,重点介绍了如何把原子力显微镜(AFM)成像及单分子力谱结合(包括原位结合或者离位结合)起来,研究高分子之间的相互作用.本文涉及生物高分子(主要是核酸-蛋白质体系)以及合成高分子体系(如聚氧乙烯,PEO)的相关研究工作.对于生物高分子体系,主要以长链核酸(如双螺旋DNA及RNA)为探针,首先利用A...     

Force measurements of bacterial adhesion on metals using a cell probe atomic force microscope

The adhesion of microbial cells to metal surfaces in aqueous media is an important phenomenon in both the natural environment and engineering systems. The adhesion of two anaerobic sulfate-reducing bacteria (Desulfovibrio desulfuricans and a local marine isolate) and an aerobe (Pseudomonas sp.) to four polished metal surfaces (i.e., stainless steel 316, mild steel, aluminum, and copper) was examined using a force spectroscopy technique with an atomic force microscope (AFM). Using a modified bacterial tip, the attraction and repulsion forces (in the nano-Newton range) between the bacterial cell and the metal surface in aqueous media were quantified. Results show that the bacterial adhesion force to aluminum is the highest among the metals investigated, whereas the one to copper is the lowest. The bacterial adhesion forces to metals are influenced by both the electrostatic force and metal surface hydrophobicity. It is also found that the physiological properties of the bacterium, namely the bacterial surface charges and hydrophobicity, also have influence on the bacteria-metal interaction. The adhesion to the metals by Pseudomonas sp. and D. desulfuricans was greater than by the marine SRB isolate. The cell-cell interactions show that there are strong electrostatic repulsion forces between bacterial cells. Cell probe atomic force microscopy has provided some useful insight into the interactions of bacterial cells with the metal surfaces.     

Estimating the thickness of hydrated ultrathin poly(o-phenylenediamine) film by atomic force microscopy

A novel method to measure ultrathin poly(o-phenylenediamine) (PPD) film electropolymerized on gold electrode in liquid was developed. It is based on the force versus distance curve (force curve) of atomic force microscopy (AFM). When 1-0.25 μm/s was chosen as the rising rate of the scanner, and 50% of the confidence interval (CI) as the qualifying threshold value, the thickness of the hydrated polymer film could be calculated. This result was compared with one obtained from an AFM image. A step-like electrode fabricated by a photolithographic process was used. The height difference of the electrode before and after the PPD coating was imaged in liquid, and then the real thickness, 19.6±5.2 nm, was obtained. The sample was also measured by estimating the transition range of the force curve of hydrated PPD film, and the thickness of the hydrated PPD film was determined to be 19.3±8.2 nm. However, the results calculated by integrating the electropolymerized charge for the oxidation process of o-phenylenediamine (o-PD) was only one-third as large as it was when using the two previously described methods. This indicated that the structure of hydrated PPD film might have been swollen.     

Spectral force analysis using atomic force microscopy reveals the importance of surface heterogeneity in bacterial and colloid adhesion to engineered surfaces

Coatings developed to reduce biofouling of engineered surfaces do not always perform as expected based on their native properties. One reason is that a relatively small number of highly adhesive sites, or the heterogeneity of the coated surface, may control the overall response of the system to initial bacterial deposition. It is shown here using an approach we call spectral force analysis (SFA), based on force volume imaging of the surface with atomic force microscopy, that the behavior of surfaces and coatings can be better understood relative to bacterial adhesion. The application of vapor deposited TiO₂ metal oxide increased bacterial and colloid adhesion, but coating the surface with silica oxide reduced adhesion in a manner consistent with SFA based on analysis of the “stickiest” sites. Application of a TiO₂-based paint to a surface produced a relatively non-fouling surface. Addition of a hydrophilic layer coating to this surface should have decreased fouling. However, it was observed that this coating actually increased fouling. Using SFA it was shown that the reason for the increased adhesion of bacteria and particles to the hydrophilic layer was that the surface produced by this coating was highly heterogeneous, resulting in a small number of sites that created a stickier surface. These results show that while it is important to manufacture surfaces with coatings that are relatively non-adhesive to bacteria, it is also essential that these coatings have a highly uniform surface chemistry.     

Investigation on the folding mode of a polymer chain in a spiral crystal by single molecule force spectroscopy

Investigation on the folding mode of a single polymer chain in its crystal is significant to the understanding of the mechanism of the fundamental crystallization as well as the engineering of new polymer crystal-based materials. Herein, we use the combined techniques of atomic force microscopy （AFM） imaging and force spectroscopy to pull a single polyethylene oxide （PEO） chain out of its spiral crystal in amyl acetate. From these data, the folding mode of polymer chains in the spiral crystal has been reconstructed. We find that the stems tilt in the typical flat area, leading to the decrease in the apparent lamellar height. While in the area of screw dislocation, the lamellar height gradually increases in the range of several nanometers. These results indicate that the combined techniques present a novel tool to directly unravel the chain folding mode of spiral crystals at single-molecule level.     

Interaction between solid surfaces in a polymer melt studied by atomic force microscopy

Using an atomic force microscope (AFM) the interaction between an AFM tip and a planar silicon oxide surface has been measured across poly(dimethylsiloxane) (PDMS, M_W = 18 000). Due to the small radius of curvature of the AFM tip the hydrodynamic repulsion of the tip was negligible and forces could be measured in equilibrium. This is confirmed by the fact that force-versus-distance curves measured at different approaching velocities were indistinguishable. In equilibrium a repulsive force was observed which could best be described by a power law, F ∝ 1/d^2.5 where d is the distance.     

Atomic force microscopy of soil and stream fulvic acids

Atomic force microscopy (AFM) was used to image fulvic acid (FA) deposited from aqueous solution on to the basal-plane surfaces of freshly cleaved muscovite, and allowed to air dry. Two fulvic acid samples were used: a soil fulvic acid (SFA) prepared by NaOH extraction from a muck soil underlying a freshwater fen in the New Jersey Pinelands and the IHSS standard Suwannee River fulvic acid (SRFA). The use of tapping-mode AFM (TMAFM), a relatively new technique which reduces the lateral frictional forces generally associated with contact-mode AFM, allowed excellent images of delicate FA structures to be obtained with minimal sample disturbance. Four main structures were observed on SFA. At low concentrations, sponge-like structures consisting of rings ( 15 nm in diameter) appeared, along with small spheres (10–50 nm). At higher concentrations, aggregates of spheres formed branches and chain-like assemblies. At very high surface coverage, perforated sheets were observed. On some samples, all of these structures were apparent, perhaps owing to concentration gradients on drying. SRFA samples were only imagined at higher concentrations. Spheres, aggregated branches, and perforated sheets were apparent. The results agree with previous work by Stevenson and Schnitzer [Soil Sci., 133(1992) 179], who applied TEM to soil FAs freeze-dried on muscovite. However, the TEM images did not detect the smaller spheres and sponge-like structures observed by AFM at low concentrations. The relevance of imaging dried samples remains questionable; hence, it is hoped in the future to use new in situ TMAFM to image FAs sorbed to surfaces in solution. Although TMAFM provided excellent images, a variety of artifacts and potential problems were encountered, as discussed.     

Mapping molecular adhesion sites inside SMIL coated capillaries using atomic force microscopy recognition imaging

Capillary zone electrophoresis (CZE) is a powerful analytical technique for fast and efficient separation of different analytes ranging from small inorganic ions to large proteins. However electrophoretic resolution significantly depends on the coating of the inner capillary surface. High technical efforts like Successive Multiple Ionic Polymer Layer (SMIL) generation have been taken to develop stable coatings with switchable surface charges fulfilling the requirements needed for optimal separation. Although the performance can be easily proven in normalized test runs, characterization of the coating itself remains challenging. Atomic force microscopy (AFM) allows for topographical investigation of biological and analytical relevant surfaces with nanometer resolution and yields information about the surface roughness and homogeneity. Upgrading the scanning tip to a molecular biosensor by adhesive molecules (like partly inverted charged molecules) allows for performing topography and recognition imaging (TREC). As a result, simultaneously acquired sample topography and adhesion maps can be recorded. We optimized this technique for electrophoresis capillaries and investigated the charge distribution of differently composed and treated SMIL coatings. By using the positively charged protein avidin as a single molecule sensor, we compared these SMIL coatings with respect to negative charges, resulting in adhesion maps with nanometer resolution. The capability of TREC as a functional investigation technique at the nanoscale was successfully demonstrated.     

液体环境单分子免疫球蛋白G的原子力显微镜高分辨成像

原子力显微镜技术( AFM)具有纳米级高分辨成像能力,是研究生物大分子结构和功能的重要工具之一。制备合适的样品是获取高分辨成像的关键要素。本研究结合DNA折纸技术,将抗原分子修饰在DNA折纸上,通过分子识别作用,抗体分子与抗原分子特异性结合,形成由DNA折纸和抗原抗体复合物构成的纳米结构。利用DNA折纸在云母表面上的吸附特点,使得抗体分子选择性地吸附在衬底表面上,由此获得了液体环境中的单个地高辛抗体免疫球蛋白G( IgG)分子的“Y”超微结构形貌。本方法简单、方便,为AFM在单分子水平上检测和表征生物分子结构和功能提供帮助。     

Novel recognition mechanisms in biological adhesion

Molecular recognition is generally thought of in terms of bond formation between unique sites on host and complementary guest molecules, but recent studies have revealed new complexities in biological recognition at cell. Adhesion between cell surfaces similarly involves specific interactions between analogous ligands and receptors. Recent force measurements, however, suggest that cell adhesion proteins may bind via multiple interaction sites that can form in a sequential manner. Other studies further show that in some instances, the principal recognition event may not be receptor–ligand docking, but the assembly of a complex pattern of many receptors and ligands.     

A method for visualization of biomolecules labeled by a single quantum dot in living cells by a combination of total internal reflection fluorescence microscopy and intracellular fluorescence microscopy

We developed a simple fluorescence microscopy for acquisition of high-resolution images of single quantum dots (QDs) labeled to biomolecules on apical plasma membrane, in cell interior and on basal plasma membrane of living cells. The method was a combination of total internal reflection fluorescence microscopy (TIRFM) at apical cell surface and intracellular microscopy coupled with focusing objective. Insulin conjugated to single QD (insulin-QD) was chosen as the model system. In order to bind insulin-QDs to insulin receptors on the plasma membrane through the interaction between insulin and its receptor, as well as internalize them, the cells attached on a coverslip were incubated with biotinylated insulin and QD-streptavidin conjugate at 37 °C. Next, fluorescent molecules in the cells were photobleached by illuminating the cells using a 100-W mercury lamp with the wavelengths from 460 to 490 nm. Then, the incident angle of a laser beam was adjusted to produce total internal reflection at the apical surface of a single cell. In this case, the insulin-QDs in the whole cell were excited, and the fluorescent molecules outside the cell were not illuminated. Finally, the images of single insulin-QDs on the apical plasma membrane, in the cell interior and on the basal plasma membrane of the cell were taken by focusing the objective to different positions, respectively. The resolution and contrast of the fluorescent spots in the images were much higher than those obtained by using epi-fluorescence microscopy and comparable to those obtained by using the conventional TIRFM. The method improved the image acquisition speed for the images on the apical and basal plasma membrane using the conventional TIRFM, and could acquire the high-resolution images in the cell interior quickly.     

Effect of alkaline treatment on cellulose supramolecular structure studied with combined confocal Raman spectroscopy and atomic force microscopy

The aim of this work was to investigate the morphological and structural changes associated with mercerization of cellulose fibres with combined confocal Raman and atomic force microscopy (AFM). During mercerization the alkali induces a change in polymorphic lattice from cellulose I to II. This was observed by confocal Raman spectroscopy from cellulose samples treated with 10, 15 and 25% aqueous sodium hydroxide solution. AFM images from the same samples illustrated that microfibrils were swollen and more granular in cellulose II than in cellulose I. Raman spectral images in plane and depth directions showed that the polymorphous cellulose structure was uniform throughout the cell wall, whereas the microfibril orientation varied between fibre cell wall layers. The changes in microfibril orientation on the sample surfaces were confirmed by AFM images measured from the same sample position.     

Analytical descriptions of dynamic force spectroscopy: behaviour of multiple connections

Exploration of the stressed lifetime of a single bond can reveal details of hidden transition states along the unbonding coordinate [Faraday Discuss. 111 (1998) 1]. Such experiments with single molecules are, however, not easy. To measure the force between two molecules requires manipulation of the contact so that two and only two molecules interact. This is achieved by reducing the probability of bond formation on contact through the control of surface chemistry, molecular density, contact force and time. When the contact area and surface chemistry cannot be controlled multiple interactions may dominate. The fundamental question arises whether quantitative information pertinent to the single interaction can be extracted from measurements of multiple simultaneous detachments. Various statistical methods have been adopted in an attempt to elucidate the single-molecule event from the rupture of multiple attachments [Biophys. J. 70 (5) (1996) 2437; Biochemistry 36 (24) (1997) 7457; Langmuir 12 (5) (1996) 1291]. Here, I aim to qualify and validate, if possible, such approaches. Whilst the analysis shows that the dynamics of loading multiple attachments precludes an accurate inference of the single unbinding event, the complexity in behaviour could be exploited to construct materials with novel dynamic mechanical properties.     

Intercalation-induced changes in DNA supercoiling observed in real-time by atomic force microscopy

Atomic force microscopy (AFM) has been employed to observe in real-time and in an aqueous environment the process of ethidium bromide induced supercoiling in individual DNA plasmid molecules. Image data reveal both the onset and the progressive presence of plectonemic DNA supercoiling. In addition, significant molecular motion of the surface adsorbed DNA is observed. These data illustrate the potential of AFM in the time-resolved study of biomolecular processes, and hence, provide new insights into biomolecular structure and function.     

Characterization of human cells exposed to deltamethrin by means of Raman microspectroscopy and atomic force microscopy

Raman spectroscopy is a powerful technique for studying cellular biochemistry. In fact, each toxic chemical induces biochemical changes related to the own action mechanism. In this investigation Raman microspectroscopy has been used, in correlation with atomic force microscopy images, to detect biochemical and structural damages occurring in cultured human cells as a consequence of deltamethrin exposure. Cultured human keratinocyte cells have been exposed at increasing concentrations of deltamethrin from 10⁻³ M to 10⁻⁶ M for 24 h. A viability test indicated that the cytotoxic dose corresponds to exposure at deltamethrin solution for 24 h with the chemical concentration between 10⁻⁴ M and 2.5 10⁻⁴ M. The compared analysis of Raman spectra and AFM images allows to state that an evident damage occurs in the plasmatic membrane and it is already detectable after exposure of keratinocytes at the lowest investigated deltamethrin concentration (10⁻⁶ M). The most important modifications are related to the breakdown of CH₂ bonds of lipidic chains, whereas proteineous bonds are less involved in the deltamethrin action. On the whole, cellular damage starts after exposure to deltamethrin doses well lower than that established as cytotoxic.     

Label-free quantification of Tacrolimus in biological samples by atomic force microscopy

In the present paper we describe an atomic force microscopy (AFM)-based method for the quantitative analysis of FK506 (Tacrolimus) in whole blood (WB) samples. Current reference methods used to quantify this immunosuppressive drug are based on mass spectrometry. In addition, an immunoenzymatic assay (ELISA) has been developed and is widely used in clinic, even though it shows a small but consistent overestimation of the actual drug concentration when compared with the mass spectrometry method. The AFM biosensor presented herein utilises the endogen drug receptor, FKBP12, to quantify Tacrolimus levels. The biosensor was first assayed to detect the free drug in solution, and subsequently used for the detection of Tacrolimus in blood samples. The sensor was suitable to generate a dose–response curve in the full range of clinical drug monitoring. A comparison with the clinically tested ELISA assay is also reported.