原子力显微镜在植物学研究中的应用

原子力显微镜（AFM）是一种探测样品表面信息的有力工具,它可以在空气和接近样品生理条件下成像,同时也可以在皮牛（pico-Newton,10^-12N）至微牛（micro—Newton,10^-6N）水平上测量力的大小。本文主要介绍了自AFM发明以来,其在植物大分子、细胞器、细胞、叶片等方面的应用,并列举了目前AFM存在的几点不足。     

用原子力显微技术研究受体-配体间的相互作用

原子力显微镜(AFM)不仅能对纳米生物结构进行实时动态的形态和结构观察,而且还能以10^-12N(pN)的精度对溶液中生物分子表面的相互作用力进行直接测量,逐渐成为一种研究受体-配体间相互作用的良好工具。本简要综述用AFM研究受体-配体间作用力、受体-配体间相互作用的影响因素及对这些因素的处理方法。     

原子力显微镜 (AFM) 在细菌生物被膜研究中的应用

原子力显微镜(AFM)作为一项重要的表面可视化技术,以其独特的优势(纳米级的空间分辨率、皮牛级力灵敏度、免标记、可在溶液环境下工作)被广泛应用于生物被膜的研究。AFM不仅可以在近生理环境下对生物被膜表面超微形貌进行可视化表征,同时还可以通过纳米压痕对生物被膜的机械特性(弹性和粘性)进行定量测量,利用AFM单细胞和单分子力谱技术可以获得生物被膜形成过程中细胞-基底以及细胞-细胞之间的相互作用力,为生物被膜的实时原位系统研究提供了可行性。本文简述了AFM的基本操作原理,综述了近年来AFM用于生物被膜表面超微结构成像、机械特性测量以及相互作用力研究方面的进展,并对AFM在生物被膜研究中面临的问题和未来的发展方向进行了讨论。     

利用原子力显微镜对A型流感病毒的形态学研究

利用透射电子显微镜(TEM)和原子力显微镜(AFM)观察流感病毒(H1N1),探讨AFM在病毒形态研究中的应用,为病毒形态学研究提供一种新型、简便、快捷的工具.TEM采用磷钨酸负染方法,AFM采用轻敲模式在大气常温下扫描成像,并对主要指标长度(直径)、Ra、Rq等进行测量.两种方法最终得到相似的形态学结果,流感病毒呈球状、丝状,并有一些形状介于两者之间.TEM提供了流感病毒二维图像,可见钉状突起,AFM则呈现了流感病毒三维图像,且可见病毒表面有凹凸不平的特征和边缘有齿轮状的突起,同时获得表面粗糙度等可以量化指标.与TEM观察相比,原子力显微镜是一种制样简单、观察直观的新型病毒形态学研究工具,其表征参数可以作为病毒形态学研究的量化指标.     

单个腺病毒颗粒的原子力显微镜液相成像及样品制备方法

原子力显微镜(Atomic force microscopy,AFM)作为一种纳米级高分辨率的探针扫描显微镜,在病毒形态学研究等领域有广泛的应用。为更好地利用AFM在接近病毒生理环境(液相)中观测单个病毒颗粒,选择合适的基底吸附病毒样品是重要的前提保障。本文介绍了一种病毒样品制备及AFM液相成像方法。首先制备疏水化处理的玻璃盖玻片作为吸附基底,再将观察样品腺病毒重组载体颗粒(Adenovirus type 5F35,Ad5F35)吸附于基底上,建立在液相下单个病毒颗粒的原子力显微镜成像方法。通过该方法获取了单个腺病毒颗粒在液相下的高分辨率纳米级高度图(height image),且病毒颗粒保持了约90nm的正确测量高度。实验结果表明疏水化处理的玻璃基底具有良好的吸附力,有助于病毒颗粒牢固地吸附在基底上,且不会引起较大的形变。本研究为AFM观测病毒等生物样品提供了一种较为合适的吸附基底和在液相中的AFM成像方法,对后期开展病毒形态结构、物理属性及其与宿主细胞受体相互作用的研究打下基础。     

难溶性药用纳米微粒的原子力显微检测

建立用原子力显微镜(AFM)检测难溶性药用纳米微粒的方法。将纳米微粒配成一定浓度的悬浮液,以新鲜裂解的云母表面作为样品载体,在室温下用AFM的接触模式成像,用粒度分析软件进行粒度分析。以此方法对两种微粒(活性炭及氧化锌纳米微粒)进行了成像和测量,同时对两种样品进行透射电镜(TEM)观察。AFM检测结果显示,纳米活性炭微粒形态近似球形,表面较平滑,平均直径为(299±187)nm;纳米氧化锌微粒呈球形,平均直径(50±20)nm,与TEM检测结果具有较高一致性。该法简便可靠。     

原子力显微镜在生物领域中的应用

原子力显微镜(AFM)作为生物样品表面表征的有力工具, 具有独特的优势。本文在介绍原子力显微镜基本原理的基础上, 综述了原子力显微镜样品制备以及原子力显微镜形貌分析、力曲线以及动力学分析在生物领域中的应用。     

原子力显微镜在生物医学上的应用

原子力显微镜(atomic force microscope,AFM)是扫描探针显微镜(SPM)的一种,其分辨率达到纳米级,能对从原子到分子尺度的结构进行三维成像和测量,能观察任何活的生命样品及动态过程。本文概述了AFM的基本工作原理及在生物医学上对DNA、蛋白质、细胞及生物过程等方面进行的研究。     

原子力显微镜在生物领域中的应用

原子力显微镜(AFM)作为生物样品表面表征的有力工具,具有独特的优势.本文在介绍原子力显微镜基本原理的基础上,综述了原子力显微镜样品制备以及原子力显微镜形貌分析、力曲线以及动力学分析在生物领域中的应用.     

界面上配基种类对BSA吸附行为的影响

利用双偏振极化干涉测量仪(DPI)研究了界面上配基种类对BSA吸附行为的影响。采用3-氨基丙基三乙氧基硅烷(APTES)、3-(甲氨基)丙基三甲氧基硅烷(MAPTMS)和N,N-二乙基-3-氨基丙基三甲氧基硅烷(DAPTMS)对DPI芯片进行了修饰,利用X射线光电子能谱比较了芯片上不同配基的密度,采用原子力显微镜(AFM)和DPI对界面上BSA吸附行为进行了研究。结果表明APTES修饰界面上BSA呈饼状,高配基密度易导致BSA多位点吸附。相同偶联密度条件下BSA在DAPTMS修饰芯片的吸附量高于MAPTMS修饰芯片,但吸附层厚度一致,表明DAPTMS表面上BSA存在聚集现象;AFM扫描结果与DPI分析结果一致,证明了配基密度和种类不仅影响界面上蛋白质吸附量,而且影响蛋白质吸附密度和表面聚集行为。     

Imaging the ordered arrays of water-soluble protein ferritin with the atomic force microscope

Individual water-soluble molecules of the protein ferritin have been imaged on a silicon surface in pure water at room temperature with the atomic force microscope (AFM). The ferritin molecules formed an ordered monolayer by binding to a charged polypeptide monolayer of poly-1-benzyl-L-histidine (PBLH) spread at the air-water interface. The film, fully wetted with water, was horizontally transferred onto an alkylated silicon wafer for AFM imagings. The hexagonal arrangement of ferritin molecules was imaged with high reproducibility on the whole surface of the film, since the forces between cantilever and the sample could be kept sufficiently smaller than 10^-10 N, mainly due to a “self-screening effect” of the surface charges of the ferritin-PBLH layer. This is the first observation of two-dimensional ordered arrays of water-soluble protein molecules directly confirmed by AFM with molecular resolution.     

Lateral mechanical coupling of stereocilia in cochlear hair bundles

For understanding the gating process of transduction channels in the inner ear it is essential to characterize and examine the functional properties of the ultrastructure of stereociliary bundles. There is strong evidence that transduction channels in hair cells are gated by directly pulling at the so-called tip links. In addition to these tip links a second class of filamentous structures was identified in the scanning and transmission electron microscope: the side-to-side links. These links laterally connect stereocilia of the same row of a hair bundle. This study concentrates on mechanical coupling of stereocilia of the tallest row connected by side-to-side links. Atomic Force microscopy (AFM) was used to investigate hair bundles of outer hair cells (OHCs) from postnatal rats (day 4). Although hair bundles of postnatal rats are still immature at day 4 and interconnecting cross-links do not show preferential direction yet, hair bundles of investigated OHCs already showed the characteristic V-shape of mature hair cells. In a first experiment, the stiffness of stereocilia was investigated scanning individual stereocilia with an AFM tip. The spring constant for the excitatory direction was 2.5 +/- 0.6 x 10(-3) N/m whereas a higher spring constant (3.1 +/- 1.5 x 10(-3) N/m) was observed in the inhibitory direction. In a second set of experiments, the force transmission between stereocilia of the tallest row was measured using AFM in combination with a thin glass fiber. This fiber locally displaced a stereocilium while the force laterally transmitted to the neighboring untouched taller stereocilia was measured by AFM. The results show a weak force interaction between tallest stereocilia of postnatal rats. The force exerted to an individual stereocilium declines to 36% at the nearest adjacent stereocilium of the same row not touched with the fiber. It is suggested that the amount of force transmitted from a taller stereocilium to an adjacent one of the same row depends on the orientation of links. Maximum force transmission is expected to appear along the axis of interconnecting side links. In our studies it is suggested that transmitted forces are small because connecting side links are oriented very close to an angle of 90 degrees with respect of the scan direction (excitatory-inhibitory direction).     

Assessment of elasticity and topography of Aspergillus nidulans spores via atomic force microscopy

Previous studies have described both surface morphology and adhesive properties of fungal spores, but little information is currently available on their mechanical properties. In this study, atomic force microscopy (AFM) was used to investigate both surface topography and micromechanical properties of Aspergillus nidulans spores. To assess the influence of proteins covering the spore surface, wild-type spores were compared with spores from isogenic rodA(+) and rodA(-) strains. Tapping-mode AFM images of wild-type and rodA(+) spores in air showed characteristic "rodlet" protein structures covering a granular spore surface. In comparison, rodA(-) spores were rodlet free but showed a granular surface structure similar to that of the wild-type and rodA(+) spores. Rodlets were removed from rodA(+) spores by sonication, uncovering the underlying granular layer. Both rodlet-covered and rodlet-free spores were subjected to nanoindentation measurements, conducted in air, which showed the stiffnesses to be 110 +/- 10, 120 +/- 10, and 300 +/- 20 N/m and the elastic moduli to be 6.6 +/- 0.4, 7.0 +/- 0.7, and 22 +/- 2 GPa for wild-type, rodA(+) and rodA(-) spores, respectively. These results imply the rodlet layer is significantly softer than the underlying portion of the cell wall.     

Elastic properties of the cell wall of Aspergillus nidulans studied with atomic force microscopy

Currently, little is known about the mechanical properties of filamentous fungal hyphae. To study this topic, atomic force microscopy (AFM) was used to measure cell wall mechanical properties of the model fungus Aspergillus nidulans. Wild type and a mutant strain (deltacsmA), lacking one of the chitin synthase genes, were grown in shake flasks. Hyphae were immobilized on polylysine-coated coverslips and AFM force--displacement curves were collected. When grown in complete medium, wild-type hyphae had a cell wall spring constant of 0.29 +/- 0.02 N/m. When wild-type and mutant hyphae were grown in the same medium with added KCl (0.6 M), hyphae were significantly less rigid with spring constants of 0.17 +/- 0.01 and 0.18 +/- 0.02 N/m, respectively. Electron microscopy was used to measure the cell wall thickness and hyphal radius. By use of finite element analysis (FEMLAB v 3.0, Burlington, MA) to simulate AFM indentation, the elastic modulus of wild-type hyphae grown in complete medium was determined to be 110 +/- 10 MPa. This decreased to 64 +/- 4 MPa for hyphae grown in 0.6 M KCl, implying growth medium osmotic conditions have significant effects on cell wall elasticity. Mutant hyphae grown in KCl-supplemented medium were found to have an elastic modulus of 67 +/- 6 MPa. These values are comparable with other microbial systems (e.g., yeast and bacteria). It was also found that under these growth conditions axial variation in elastic modulus along fungal hyphae was small. To determine the relationship between composition and mechanical properties, cell wall composition was measured by anion-exchange liquid chromatography and pulsed electrochemical detection. Results show similar composition between wild-type and mutant strains. Together, these data imply differences in mechanical properties may be dependent on varying molecular structure of hyphal cell walls as opposed to wall composition.     

Mechanical force analysis of peptide interactions using atomic force microscopy

Some peptides have previously been reported to bind low molecular weight chemicals. One such peptide with the amino acid sequence His-Ala-Ser-Tyr-Ser was selectively screened from a phage library and bound to a cationic porphyrin, 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphine (TMpyP), with a binding constant of 10(5) M(-1) (J. Kawakami, T. Kitano, and N. Sugimoto, Chemical Communications, 1999, pp. 1765-1766). The proposed binding was due to pi-electron stacking from two aromatic amino acids of histidine and tyrosine. In this study, the weak interactions between TMpyP and the peptide were further investigated by force curve analysis using atomic force microscopy (AFM). The mechanical force required to unbind the peptide-porphyrin complex was measured by vertical movement of the AFM tip. Peptide self-assembled monolayers were formed on both a gold-coated mica substrate and a gold-coated AFM tip. The TMpyPs could bind between the two peptide layers when the peptide-immobilized AFM tip contacted the peptide-immobilized substrate in solution containing TMpyP. In the retracting process a force that ruptured the interaction between TMpyPs and peptides was observed. The unbinding force values correlated to the concentration of TMpyP. A detection limit of 100 ng/mL porphyrin was obtained for the force measurement, and was similar to surface plasmon resonance sensor detection limits. Furthermore, we calculated the product of the observed force and the length of the molecular elongation to determine the work required to unbind the complexes. The obtained values of unbinding work were in a reasonable range compared to the binding energy of porphyrin-peptide.     

Disposable biosensor based on graphene oxide conjugated with tyrosinase assembled gold nanoparticles

A highly efficient enzyme-based screen printed electrode (SPE) was obtained by using covalent attachment between 1-pyrenebutanoic acid, succinimidyl ester (PASE) adsorbing on the graphene oxide (GO) sheets and amines of tyrosinase-protected gold nanoparticles (Tyr-Au). Herein, the bi-functional molecule PASE was assembled onto GO sheets. Subsequently, the Tyr-Au was immobilized on the PASE-GO sheets forming a biocompatible nanocomposite, which was further coated onto the working electrode surface of the SPE. The characterization of obtained nanocomposite and modified SPE surface was investigated by atomic force microscopy (AFM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Attributing to the synergistic effect of GO-Au integration and the good biocompatibility of the hybrid-material, the fabricated disposable biosensor (Tyr-Au/PASE-GO/SPE) exhibited a rapid amperometric response (less than 6s) with a high sensitivity and good storage stability for monitoring catechol. This method shows a good linearity in the range from 8.3×10(-8) to 2.3×10(-5) M for catechol with a squared correlation coefficient of 0.9980, a quantitation limit of 8.2×10(-8) M (S/N=10) and a detection limit of 2.4×10(-8) M (S/N=3). The Michaelis-Menten constant was measured to be 0.027 mM. This disposable tyrosinase biosensor could offer a great potential for rapid, cost-effective and on-field analysis of phenolic compounds.     

Unfolding and extraction of a transmembrane alpha-helical peptide: dynamic force spectroscopy and molecular dynamics simulations

An atomic force microscope (AFM) was used to visualize CWALP(19)23 peptides ((+)H(3)N-ACAGAWWLALALALALALALWWA-COO(-)) inserted in gel-phase DPPC and DSPC bilayers. The peptides assemble in stable linear structures and domains. A model for the organization of the peptides is given from AFM images and a 20 ns molecular dynamics (MD) simulation. Gold-coated AFM cantilevers were used to extract single peptides from the bilayer through covalent bonding to the cystein residue. Experimental and simulated force curves show two distinct force maxima. In the simulations these two maxima correspond to the extraction of the two pairs of tryptophan residues from the membrane. Unfolding of the peptide precedes extraction of the second distal set of tryptophans. To probe the energies involved, AFM force curves were obtained from 10 to 10(4) nm/s and MD force curves were simulated with 10(8)-10(11) nm/s pulling velocities (V). The velocity relationship with the force, F, was fitted to two fluctuation adhesive potential models. The first assumes the pulling produces a constant bias in the potential and predicts an F approximately ln (V) relationship. The second takes into account the ramped bias that the linker feels as it is being driven out of the adhesion complex and scales as F approximately (ln V)2/3.     

Electrochemical selective determination of ascorbic acid at redox active polymer modified electrode derived from direct blue 71

A simple selective method for determination of ascorbic acid using polymerized direct blue 71 (DB71) is described. Anodic polymerization of the azo dye DB71 on glassy carbon (GC) electrode in 0.1M H(2)SO(4) acidic medium was found to yield thin and stable polymeric films. The poly(DB71) films were electroactive in wide pH range (1-13). A pair of symmetrical redox peaks at a formal redox potential, E('0)=-0.02V vs. Ag/AgCl (pH 7.0) was observed with a Nernstian slope -0.058V, is attributed to a 1:1 proton+electron involving polymer redox reactions at the modified electrode. Scanning electron microscope (SEM), atomic force microscope (AFM) and electrochemical impedance spectroscopy (EIS) measurements were used for surface studies of polymer modified electrode. Poly(DB71) modified GC electrode showed excellent electrocatalytic activity towards ascorbic acid in neutral buffer solution. Using amperometric method, linear range (1x10(-6)-2x10(-3)M), dynamic range (1x10(-6)-0.01M) and detection limit (1x10(-6)M, S/N=3) were estimated for measurement of ascorbic acid in pH 7.0 buffer solution. Major interferences such as dopamine and uric acid are tested at this modified electrode and found that selective detection of ascorbic acid can be achieved. This new method successfully applied for determination of ascorbic acid in commercial tablets with satisfactory results.