光学分子成像技术观察纳流芯片对DNA分子的研究

λ-DNA与荧光染料YOYO-1结合,利用荧光显微技术对DNA分子在毛细现象作用下进入宽40 nm、深60 nm的纳米沟道内,并在其内部被拉伸以及沿沟道移动的情形进行了观察.讨论了DNA分子在沟道内的运动情况.结合光学分子成像技术与该尺寸范围的纳流芯片,将有助于研究生物分子的动力学和静力学性质.     

拉直的单个DNA分子的全内反射荧光实时成像研究

全内反射荧光(TIRF)成像技术利用穿透深度仅200 nm左右的隐失波来激发诱导荧光,探测灵敏度和图像信噪比大大提高,成为单分子研究的有力工具。分子梳技术利用DNA末端与固体表面的结合力和周围流体流动产生的侧向力将DNA分子拉伸并平铺在表面上。结合这两种技术,对分子梳拉直的单个DNA分子进行了清晰的实时荧光成像,发现TIRF成像条件下DNA分子与荧光探针YOYO-1组成的复合体可自然避免发生光敏断裂现象;实时监测了单个DNA-YOYO-1复合体的光漂白过程,通过对激发光照射时间与探测器曝光时间进行同步控制,可大幅降低光漂白程度,为拉直的单个DNA分子的长时间实时观察和成像研究优化了实验条件,为实时、可视化地研究其与蛋白质相互作用的动力学过程奠定了基础。     

Photo-induced synthesis of DNA-templated metallic nanowires and their integration into micro-fabricated contact arrays

We report the synthesis of metallic nanowires accomplished by site-specific integration of single DNA duplexes into micro-fabricated contact arrays and their subsequent selective metallization. DNA interconnects between metallic contacts are formed by tethering the ends of DNA molecules, stretched in hydrodynamic flow into a linear conformation, at different gold contact pads via thiol functional groups. To transform the DNA interconnects into metallic cluster chains or nanowires, we use an electroless metal deposition technique where platinum ions bound along the DNA molecules from a salt solution are reduced to metallic clusters of less than 10 nm in diameter by applying UV light.     

Surface-enhanced Raman scattering of λ -DNA

By using the molecular combing method, we stretched DNA molecules with colloidal silver nanoparticles adsorbed on them. Surface-enhanced Raman scattering (SERS) spectra were recorded in this particular DNA/Ag system. We also studied SERS spectra of DNA molecules in solution. The spectra of DNA in different concentration solution show distinct differences, and we speculate that the coiling states of DNA molecules are different in different concentrations, leading to the spectral differences. PACS 33.20.Fb; 42.62.Fi; 78.67.-n; 73.20.Mf; 87.64.-t     

Metallization of DNA on silicon surface

New simple way for silver deoxyribonucleic acid (DNA)-based nanowires preparation on silicon surface was developed. The electrochemical reduction of silver ions fixed on DNA molecule provides the forming of tightly matched zonate silver clusters. Highly homogeneous metallic clusters have a size about 30 nm. So the thickness of nanowires does not exceed 30–50 nm. The surface of n-type silicon monocrystal is the most convenient substrate for this procedure. The comparative analysis of DNA metallization on of n-type silicon with a similar way for nanowires fabrication on p-type silicon, freshly cleaved mica, and glass surface shows the advantage of n-type silicon, which is not only the substrate for DNA fixation but also the source of electrons for silver reduction. Images of bound DNA molecules and fabricated nanowires have been obtained using an atomic force microscope and a scanning ion helium microscope. DNA interaction with silver ions in a solution was examined by the methods of ultraviolet spectroscopy and circular dichroism.     

Coulomb forces on DNA polymers in charged fluidic nanoslits

We investigate the repulsive electrostatic interactions between a DNA polyelectrolyte and the charged walls of a fluidic nanoslit. The scaling of the DNA coil size with the physical slit height revealed electrostatic depletion regions that reduced the effective slit height. These regions exceeded the Debye screening length of the buffer, λ(D)(buffer), and saturated at ≈ 50 nm when λ(D)(buffer) reached 10 nm. We explain these results by modeling a semiflexible charged rod near a charged wall and the electrostatic screening by the polyelectrolyte. These results demonstrate the surprisingly long range over which a nanofluidic device can exert field-effect control over confined molecules.     

Quasi-nanowires from fluorescent semiconductor nanocrystals on the surface of oriented DNA molecules

Ordered structures in the form of quasi-nanowires were obtained from CdSe/ZnS fluorescent semiconductor nanoparticles of spherical (quantum dots) or rodlike (quantum rods) form by their electrostatic deposition on DNA molecules with subsequent stretching of the molecules on a solid substrate. Positively charged nanoparticles were fixed along the negatively charged backbones of DNA molecules by electrostatic interactions in an aqueous solution of a mixture of DNA with quantum particles at different stoichiometric ratios. Strands of single DNA molecules with quantum particles fixed along them were immobilized and stretched on hydrophobic surfaces using the molecular combing technique. It is shown that, by varying the nanoparticle charge and the stoichiometry of complexes of DNA with particles, it is possible to create fluorescent structures with predetermined morphology and properties.     

Characterization of microstructures induced in the workpiece of aluminum alloy by excimer laser micromachining

Excimer laser emitting at 248 nm is applied to produce microstructures on the surface of aluminum alloy. The surface morphology shows that hotspots and thermal fluidic structures both come to light. Two possible mechanisms of hotspots formation are proposed: near-field diffraction and interference, and extremely fast rapid thermal annealing. And for the formation of thermal fluidic pattern structure, a thin film model is applied.     

应用分子梳技术对DNA与组蛋白相互作用的研究

利用分子梳技术对λ DNA和组蛋白的相互作用进行了研究. 通过这种简单有效的方法，我们将λ DNA分子拉伸到26—28 μm，相当于其原长（约162 μm）的16—17倍. 当组蛋白与DNA结合后，DNA分子发生凝聚现象，复合体的拉伸长度明显变短，其峰值分布在10—14 μm之间. DNA 组蛋白复合体的拉伸长度与组蛋白的浓度、与碱基对和荧光染料的比例有显著的关系. 关键词： 分子梳 组蛋白 DNA 荧光显微     

Tuning the geometry of shape-restricted DNA molecules on the functionalized Si(1 1 1)

Designing a well-defined and stable interface between biomolecules and semiconductor surfaces is of great importance for current and future biosensing and bioelectronic applications. The well-characterized chemistry, stability, and easily tunable electronic properties of silicon substrate make it a practical platform for this type of interface. It has been established in our previous work that a robust, covalent attachment between thiol-DNA molecules of a pre-designed geometrical shape and a modified silicon surface can be achieved. This work focuses on using this binding model and altering the distance between the DNA molecules and silicon surface by strategically placing thiol linkers within the pre-determined geometric design of the rectangularly shaped DNA. The statistical analysis of the height profiles of DNA molecules attached to the surface, as determined by AFM, provides specific insight into how the construction of the DNA molecules affects the binding distance. A comparison between two thiol-DNA molecules with different numbers of thiol groups placed either within the rectangular shape or anchored to the free loop of the same geometric design suggest that the average distance of these molecules to the functionalized silicon surface can be changed by approximately 0.5 nm.     

Cylindrical microcavity laser based on the evanescent-wave-coupled gain

A microcavity laser based on the gain only in the evanescent field region of whispering gallery modes has been demonstrated. A cylindrical microcavity of 125 microm diam was surrounded by rhodamine 6G dye molecules in an ethanol solution of lower refractive index such that whispering gallery modes of the microcavity underwent laser oscillation when the dye molecules in the evanescent field region outside the cavity were excited by a second harmonic of a Nd:YAG laser. For particular pumping spots, single-mode laser oscillation of a transverse magnetic mode was observed at about 600 nm with associated cavity Q of 3x10(7).     

Dissection,acquisition and amplification of targeted position of electrostatically stretched DNA

The authors have developed a DNA sequencing method based on molecular surgery, where DNA strands are stretched under a high-intensity electrostatic field, positioned onto a solid surface by dielectrophoresis, and then a targeted position is mechanically dissected under a microscope, picked up and recovered. Because thousands of molecules are stretched and aligned with one end straight on the electrode edge, as many fragments from the same location can be dissected simultaneously. The method enables the sequencing of known position on the DNA, so tedious re-construction processes in the present shot-gun sequencing can be eliminated. It is found that the mechanically dissected fragment can be recovered without loss. However, molecular damage due to mechanical dissection prohibits the ligation of adapter oligonucleotide, which is necessary for the amplification of the acquired fragment. In this paper, we employ a method using a restriction enzyme for the removal of the damaged part on the fragments. Based on the method, we found that the yield of an adapter ligated on one end is about 30%, and on both ends is about 10%, which is adequate for the amplification and sequencing.     

Direct fabrication of physical relief structures via patterned photodeposition of a titanium alkoxide solution

Ultraviolet (lambda=248 nm) excitation of a photosensitive Ti alkoxide solution was found to generate a metal-oxide-based insoluble film on substrates in contact with the solution during illumination. Patterned deposition of 100 microm wide lines of material was demonstrated using a slit-shaped aluminum shadow mask during exposure. Stylus profilometry confirmed that the average thickness of the photodeposited film monotonically varied with accumulated UV fluence, exhibiting thicknesses of 10 to 310 nm for fluences of 12 and 192 J/cm(2), respectively. Moreover, the surface profile of the film surface at fluences greater than 12 J/cm(2) was found to reproduce the near-field Fresnel diffraction pattern anticipated from the slit mask used.     

电解法制备纳米银溶胶及其SERS活性研究

分别用柠檬酸三钠溶液、硝酸银和聚乙烯醇混合液作为电解液,用银棒作为电极,加上7 V直流电压,通电1 h,用电解方法得到了纳米银溶胶。为测试该纳米银溶胶是否具有表面增强拉曼散射1(SERS)活性,选用了阳离子型分子碱性品红(Fuchsine basic)、亚甲基蓝(Methylene blue),阴离子型分子苯甲酸(Benzoic acid),甲基橙(Methyl orange)、中性分子吖啶橙(Alcidine orange)、苏丹红(Sudan red)作为测试分子,进行SERS研究,结果发现用两种电解液制备的纳米银都具有很强的SERS活性,但用硝酸银和聚乙烯醇混合液作为电解液制备的纳米银溶胶具有更广泛的SERS 活性。在该方法制备的纳米银上,得到了在常规方法制备的胶态纳米银上及用柠檬酸三钠溶液作为电解液制备的纳米银上得不到的甲基橙分子的SERS谱,对可能的原因进行了讨论。     

聚甲基丙烯酸甲酯间隔的金纳米立方体与金膜复合结构的表面增强拉曼散射研究

金属纳米颗粒与金属薄膜的复合结构由于其局域表面等离子体和传播表面等离子体间的强共振耦合作用,可作为表面增强拉曼散射(SERS)基底,显著增强吸附分子的拉曼信号.本文提出了一种聚甲基丙烯酸甲酯(PMMA)间隔的90 nm金纳米立方体与50 nm金膜复合结构的SERS基底,通过有限元方法数值模拟,得到PMMA的最优化厚度为15 nm.实验制备了PMMA间隔层厚度为14 nm的复合结构,利用罗丹明6G (R6G)为拉曼探针分子, 633 nm的氦氖激光器作为激发光源,研究了复合结构和单一金纳米立方体的SERS效应,发现复合结构可以使探针分子产生比单一结构更强的拉曼信号.在此基础上,研究了不同浓度金纳米立方体水溶液条件下复合结构中R6G的拉曼光谱.结果表明,当金纳米立方体水溶液浓度为5.625μg/mL的条件下复合结构中R6G的拉曼信号最强,且可测量R6G的最低浓度达10~(–11) mol/L.     

Fluorescence enhancement of acridine orange in a water solution by Au nanoparticles

The surface enhanced fluorescence effect of acridine orange fluorophore in the proximity of Au nanoparticles has been investigated experimentally in the system of aqueous solution.Significant enhancement of the fluorescence intensity was observed when the system was excited with 532 nm or 442 nm CW lasers.The influence of the distances between neighboring Au particles as well as that between the fluorophore molecules and the Au surface were explored experimentally.The results demonstrated that a compact distribution of metallic particles was able to produce stronger fluorescence enhancement.Proper separation between the fluorophore molecules and the metal surface was favorable for a better enhancement.     

Direct observation of λ-DNA molecule reversal movement within microfluidic channels under electric field with single molecule imaging technique

The electrodynamic characteristics of single DNA molecules moving within micro-/nano-fluidic channels are important in the design of biomedical chips and bimolecular sensors. In this study, the dynamic properties of λ-DNA molecules transferring along the microchannels driven by the external electrickinetic force were systemically investigated with the single molecule fluorescence imaging technique. The experimental results indicated that the velocity of DNA molecules was strictly dependent on the value of the applied electric field and the diameter of the channel. The larger the external electric field, the larger the velocity, and the more significant deformation of DNA molecules. More meaningfully, it was found that the moving directions of DNA molecules had two completely different directions:(i) along the direction of the external electric field, when the electric field intensity was smaller than a certain threshold value;(ii) opposite to the direction of the external electric field, when the electric field intensity was greater than the threshold electric field intensity.The reversal movement of DNA molecules was mainly determined by the competition between the electrophoresis force and the influence of electro-osmosis flow. These new findings will theoretically guide the practical application of fluidic channel sensors and lab-on-chips for precisely manipulating single DNA molecules.     

The Effect of Physical Form of DNA on ExonucleaseIII Activity Revealed by Single-molecule Observations

Single-molecule studies have revealed molecular behaviors usually hidden in the ensemble and time averaging of bulk experiments. Single-molecule measurement that can control physical form of individual DNA molecules is a powerful method to obtain new knowledge about correlation between DNA-tension and enzyme activity. Here we study the effect of physical form of DNA on exonucleaseIII (ExoIII) reaction. ExoIII has a double-stranded DNA specific 3′→5′ exonuclease activity and the digestion is distributive. We observed the ExoIII digestion of individual stretched DNA molecules from the free ends. The sequentially captured photographs demonstrated that the digested DNA molecule linearly shortened with the reaction time. We also carried out the single-molecule observation under random coiled form by pausing the buffer flow. The digestion rates obtained from both single-molecule experiments showed that the digestion rate under the stretched condition was two times higher than the random coiled condition. The correlation between physical form of DNA and digestion rate of ExoIII was clearly demonstrated by single-molecule observations.     

Long term hydrophilic coating on poly(dimethylsiloxane) substrates for microfluidic applications

Poly(dimethylsiloxane) (PDMS) has been used extensively for microfluidic components and as substrates for biological applications. Since the native nature of PDMS is hydrophobic it requires a functionalization step for use in conjunction with aqueous media. Commonly, oxygen plasma treatment is used for the formation of hydrophilic groups on the surface. However, the hydrophilic nature of these surfaces is short lived and the surfaces quickly revert back to their original hydrophobic state. In this work, branched-polyethylenimine (b-PEI) was used for long term modification of plasma treated PDMS surface. Contact angle, X-ray photoelectron spectroscopy (XPS) and Atomic force microscopy (AFM) were used for characterization of the modified surfaces and their stability with time was studied. The results obtained demonstrate that comparatively higher stability, hydrophilic, positively charged surfaces can be obtained after b-PEI treatment. These b-PEI treated PDMS surfaces can be used as fluidic channels for the separation of molecules as well as a substrate for the adherence of bio-molecules or biological cells.     

Aggregation of a hydrophobically modified poly(propylene imine) dendrimer

The poly(propylene imine) dendrimer DAB-dendr- (NH2)8 was hydrophobically modified with dodecanoyl end groups. The modified dendrimer was deposited onto mica by adsorption from solution and observed by atomic force microscopy. With the decrease of adsorption time, the modified dendrimer varied from continuous film to scattered islands. For the adsorption time of 20s the dendrimer formed a sub-monolayer thin film that contained many fractal aggregates of fractal dimension 1.80 that were > 1 microm in diameter and no more than 0.8nm thick. After 5 months at 1#1 , the initial fractal aggregates transformed into disks and other less-branched shapes with average heights of the domains of 0.6nm and 0.4nm, respectively. Formation of the fractal aggregates is explained by diffusion-limited aggregation. The slow reorganization of dendrimer molecules in the fractal aggregates occurs at a temperature well above the Tg of the dendrimer.