Covalent attachment and hybridization of DNA oligonucleotides on patterned single-walled carbon nanotube films

DNA oligonucleotides were covalently immobilized to prepatterned single-walled carbon nanotube (SWNT) multilayer films by amidation. SWNT multilayer films were constructed via consecutive condensation reactions creating stacks of functionalized SWNT layers linked together by 4,4'-oxydianiline. Aminated- or carboxylated-DNA oligonucleotides were covalently immobilized to the respective carboxylated or aminated SWNT multilayer films through amide bond formation using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. UV-vis-NIR spectroscopic analysis indicated that the SWNT film surface density increased uniformly according to the number of reaction cycles. Scanning electron microscopy and contact angle measurements of the SWNT multilayer film revealed a uniform coverage over the substrate surface. The covalent attachment of DNA oligonucleotides to the SWNT multilayer films and their subsequent hybridization with complementary oligonucleotides were verified using X-ray photoelectron spectroscopy and fluorescence-based measurements. This is the first report demonstrating that DNA oligonucleotides can be covalently attached to immobilized SWNT multilayer films. The anchored DNA oligonucleotides were shown to exhibit excellent specificity, realizing their potential in future biosensor applications.     

Probing the dynamic nature of DNA multilayer films using förster resonance energy transfer

DNA films are of interest for use in a number of areas, including sensing, diagnostics, and as drug/gene delivery carriers. The specific base pairing of DNA materials can be used to manipulate their architecture and degradability. The programmable nature of these materials leads to complex and unexpected structures that can be formed from solution assembly. Herein, we investigate the structure of DNA multilayer films using F?rster resonance energy transfer (FRET). The DNA films are assembled on silica particles by depositing alternating layers of homopolymeric diblocks (polyA(15)G(15) and polyT(15)C(15)) with fluorophore (polyA(15)G(15)-TAMRA) and quencher (polyT(15)C(15)-BHQ2) layers incorporated at predesigned locations throughout the films. Our results show that DNA films are dynamic structures that undergo rearrangement. This occurs when the multilayer films are perturbed during new layer formation through hybridization but can also take place spontaneously when left over time. These films are anticipated to be useful in drug delivery applications and sensing applications.     

Multilayer film assembled from charged derivatives of chitosan: physical characteristics and biological responses

Polyelectrolyte multilayer films were successfully assembled from each of the three charged derivatives of chitosan; N-[(2-hydroxyl-3-trimethylammonium)propyl]chitosan chloride (HTACC), N-succinyl chitosan (SCC) and N-sulfofurfuryl chitosan (SFC), paired with one of the two oppositely charged polyelectrolytes, poly(acrylic acid) (PAA) and poly(allylamine hydrochloride) (PAH) on surface-treated poly(ethylene terephthalate) (treated PET) substrates by alternate layer-by-layer adsorption. Surface coverage and wettability of the multilayer films were determined by AFM and water contact angle measurements, respectively. Analysis by quartz crystal balance with dissipation (QCM-D) has suggested that all multilayer films are relatively rigid and have a high water content associated within their structures, accounting for up to 85-90% (w/w) for films having 7-10 layers. In vitro cytocompatibility tests for the fibroblast-like L929 cell line revealed a slight dependency for cell adhesion and proliferation on the outermost layer. The multilayer film containing HTACC exhibited moderate antibacterial activity against E. coli and S. aureus. Bearing negative charges, the multilayer films terminating with SFC and having at least 10 layers were capable of suppressing the adsorption of plasma proteins and platelet adhesion at a comparable level to the multilayer film assembled from heparin, a well-known antithrombogenic polymer.     

自组装膜中原位光反应固定化脱氧核糖核酸及膜稳定性

通过静电沉积的方法构筑了含有脱氧核糖核酸(DNA)和重氮树脂(DAR)的交替多层膜.在紫外光照射下,这种静电沉积多层膜相邻层间DNA上的磷酸基团与DAR上的重氮基团发生反应,从而将DNA共价连接到多层膜中.利用紫外-可见吸收光谱和掠角反射吸收傅里叶变换红外光谱(GRAFTIR)研究了这种相邻层间的光化学变化.刻蚀实验结果发现,与光照前相比,光照后的膜在盐溶液中的稳定性大大增强.     

Release of a dye from hydrogen-bonded and electrostatically assembled polymer films triggered by adsorption of a polyelectrolyte

The absorption of dyes within hydrogen-bonded and electrostatically assembled multilayers and subsequent release of the dyes from the films were studied in situ using FTIR-ATR. Multilayers were composed of poly(methacrylic acid), PMAA, and poly(ethylene oxide), PEO (hydrogen-bonded multilayers), or of PMAA and 22% quarternized copolymer of N-ethyl-4-vinylpyridium bromide and 4-vinylpyridine, Q22 (electrostatically stabilized multilayers). After multilayer deposition, the solution pH was changed to produce excess charge within the films. Dyes with charge opposite to the excess charge of the film (Rhodamine 6G for hydrogen-bonded films or Bromophenol Blue for electrostatically assembled multilayers) were then allowed to absorb within multilayers. In both systems, the dyes were uniformly included within the films. The top layers largely affected the loading capacity of the multilayers, suggesting weaker binding of the dyes with the top layers. Dye release into a 0.01 M phosphate buffer was significantly smaller as compared to release in the presence of 0.05-0.5 mg/mL solutions of adsorbing polymers whose charge was the same as the excess charge within the films. We found that with the PMAA/PEO films, dye release did not depend on the concentration of polymer in solution, but was largely controlled by the amount of charge accumulated within the adsorbing polymer layer on the top of the film. For electrostatically stabilized PMAA/Q22 systems, dye release increased with increasing concentration of Q22 in solution, suggesting a significant contribution of the competition of solution species in the release mechanism. Our findings contribute to the understanding of interactions of small molecules with polymer multilayers and might have ramifications for novel applications of multilayer films as new materials for the controlled delivery of chemicals.     

Stabilizing or destabilizing oligodeoxynucleotide duplexes containing single 2'-deoxyuridine residues with 5-alkynyl substituents

The 5-position of pyrimidines in DNA duplexes offers a site for introducing alkynyl substituents that protrude into the major groove and thus do not sterically interfere with helix formation. Substituents introduced at the 5-position of the deoxyuridine residue of dU:dA base pairs may stabilize duplexes and reinforce helices weakened by a low G/C content, which would otherwise lead to false negative results in DNA chip experiments. Here we report on a method for preparing oligonucleotides with a 5-alkynyl substituent at a 2'-deoxyuridine residue by on-support Sonogashira coupling involving the fully assembled oligonucleotide. A total of 25 oligonucleotides with 5-alkynyl substituents were prepared. The substituents either decrease the UV melting point of the duplex with the complementary strand or increase it by up to 7.1 degrees C, compared with that of the unmodified control duplex. The most duplex-stabilizing substituent, a pyrenylbutyramidopropyne moiety, is likely to intercalate but does not prevent sequence-specific base pairing of the modified deoxyuridine residue or the neighboring nucleotides. It also increases the signal for a target strand when employed on a small oligonucleotide microarray. The ability to tune the melting point of a DNA dodecamer duplex with a single side chain over a temperature range of >11 degrees C may prove useful when developing DNA sequences for biomedical applications.     

Induced-fit recognition of DNA by small circular oligonucleotides

We have investigated the molecular interaction between cyclic and linear oligonucleotides. We have found that short cyclic oligonucleotides can induce hairpinlike structures in linear DNA fragments. By using NMR and CD spectroscopy we have studied the interaction of the cyclic oligonucleotide d with d, as well as with its two linear analogs d(GTCCCTCA) and d(CTCAGTCC). Here we report the NMR structural study of these complexes. Recognition between these oligonucleotides occurs through formation of four intermolecular Watson-Crick base pairs. The three-dimensional structure is stabilized by two tetrads, formed by facing the minor-groove side of the Watson-Crick base pairs. Overall, the structure is similar to those observed previously in other quadruplexes formed by minor-groove alignment of Watson-Crick base pairs. However, in this case the complexes are heterodimeric and are formed by two different tetrads (G:C:A:T and G:C:G:C). These complexes represent a new model of DNA recognition by small cyclic oligonucleotides, increasing the number of potential applications of these interesting molecules.     

Investigations of Thin Films with Amphiphilic Dendrimers Bearing Peripheral Fullerene Subunits

In spite of a molecular mass of 7704.6 g mol(-1), third-generation compound G3 (shown schematically; Z=C(8)H(17)) is able to form stable Langmuir films. In a systematic study, the amphiphilic properties of the corresponding dendrimers of first (G1) and second generation (G2), with one and two peripheral fullerene units, respectively, were investigated and a model could be proposed for the multilayer films obtained from G1.     

Simultaneous direct electrochemiluminescence and catalytic voltammetry detection of DNA in ultrathin films

Direct electrochemiluminescence (ECL) involving DNA was demonstrated in 10 nm films of cationic polymer [Ru(bpy)(2)(PVP)(10)](2+) assembled layer-by-layer with DNA. A square wave voltammetric waveform oxidized the Ru(II) sites in the metallopolymer to Ru(III), and ECL was measured simultaneously with catalytic voltammetric peaks in a simple apparatus. Significant ECL generation occurred only when guanine bases were present on oligonucleotides in the films. This result along with knowledge of proposed ECL pathways suggests that guanine radicals initially formed by catalytic oxidation of guanines by Ru(III) react with the metallopolymer to produce electronically exited Ru(II) sites in the film. ECL and catalytic SWV peaks were sensitive to oligonucleotide hybridization and chemical DNA damage. Simultaneous linear growth of ECL and SWV peaks occurred after incubation with known DNA damage agent styrene oxide over 20 min. The estimated detection limit was 1 damaged DNA base in 1000. Control incubations of metallopolymer/ds-DNA films in buffer containing unreactive toluene resulted in no significant changes of the ECL or SWV peaks.     

表面活性剂胶束实现疏水分子在聚合物微凝胶层层组装膜中的高效负载

基于层层组装技术制备了聚烯丙基胺-葡聚糖微凝胶(记作PAH-D)/透明质酸钠(HA)膜, 将包覆有芘分子的十二烷基硫酸钠(SDS)表面活性剂胶束基于静电作用力负载到PAH-D/HA微凝胶膜中, 实现了疏水分子芘在微凝胶膜中的高效负载. 紫外-可见吸收光谱和荧光光谱证实了SDS胶束包覆的芘分子被稳定地负载在PAH-D微凝胶膜中. 透过光谱表明负载有芘分子的(PAH-D/HA)*10微凝胶膜在可见光区仍保持良好光学透过性. 芘在膜中的负载量可以通过改变PAH-D/HA微凝胶膜的沉积周期数和SDS胶束中包覆芘分子的浓度而实现调控. 具有光致变色性质的螺吡喃分子同样可以借助SDS胶束负载到PAH-D/HA微凝胶膜中, 制备具有光致变色性质的层层组装膜. 本工作为疏水有机分子在层层组装聚合物膜中的高效负载提供了一种简便、易行的方法.     

Buildup of ultrathin multilayer films by a self-assembly process, 1 consecutive adsorption of anionic and cationic bipolar amphiphiles on charged surfaces

An anionic and a cationic bipolar amphiphile containing rigid biphenyl cores were synthesized. The compounds were dissolved in a mixture of dimethylsulfoxide (DMSO) and water and pure water, respectively. When a solid substrate with a positively charged planar surface is immersed in the solution containing the negatively charged bipolar amphiphile, a monolayer of the amphiphile is adsorbed and due to its bipolar structure the surface charge is reversed. After rinsing in pure water the substrate is immersed in the solution containing the positively charged bipolar amphiphile. Again a monolayer is adsorbed but now the original surface charge is restored. By repeating both steps in a cyclic fashion alternating multilayer assemblies of both compounds are obtained. It is demonstrated that multilayer films, composed of at least 35 consecutively alternating layers, which corresponds to a total film thickness of 170 nm can be assembled.     

Fabrication of free-standing multilayer films by using pH-responsive microgels as sacrificial layers

A facile way to prepare free-standing polyelectrolyte multilayer films of poly(sodium 4-styrenesulfonate)(PSS)/poly(diallyldimethylammonium)(PDDA) was developed by applying a new pH-dependent sacrificial system based on cross-linked poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) microgels. The tertiary amine groups of PDMAEMA microgels can be protonated in acidic environment, and the protonated microgels were deposited by layer-by-layer (LbL) technique with PSS. PSS/PDDA multilayer films were constructed on the top of the PSS/microgels sacrificial layers. The LbL assembly process was investigated by UV–vis spectroscopy. Further study shows that the free-standing PSS/PDDA multilayer films can be obtained within 3 min by treating the as-prepared films in alkali aqueous solution with a pH of 12.0. The pH-triggered exfoliation of PSS/PDDA multilayer films provides a simple and facile way to prepare LbL assembled free-standing multilayer films.     

Sequence-specific control of azobenzene assemblies by molecular recognition of DNA

We investigated the molecular recognition between the amphiphile AzoAde, which is composed of azobenzene in the hydrophobic and adenine in the hydrophilic portion of the molecule, and oligonucleotides having a homogeneous base (dA30, dT30, dG30, and dC30) at the air-water interface. On the basis of the complementary base-pairing of DNA in the duplex, orderly arrangement of AzoAde on templated dT30 was examined using pi-A isotherm, UV-vis RAS, FT-IR RAS, and XPS measurements. Although there was little interaction between AzoAde and mismatched oligonucleotides (dA30, dG30, and dC30), AzoAde prepared on a dT30 subphase stoichiometrically assembled and interacted with dT30, subsequently forming a J-form assembly at the air-water interface. AFM observation of the LB films revealed the nanostructure of the J-formed AzoAde monolayer on the dT30 subphase as well as the domain structures of the H-formed monolayers on the other oligonucleotide subphases. Therefore, dT30 has a potential application as a template for assembling AzoAde at the air-water interface.     

Mineralization and osteoblast behavior of multilayered films on TiO2 nanotube surfaces assembled by the layer-by-layer technique

In this paper, the multilayer films of poly-L-lysine (PLL) and DNA were created on TiO₂ nanotube surfaces using the layer-by-layer (LBL) self-assembly technique. Chemical compositions of the assembled multilayered films were investigated by X-ray photoelectron spectroscopy. Biological properties of the multilayered films were evaluated by the biomimetic mineralization and osteoblast cell culture experiments. The results indicated that PLL and DNA were successfully assembled onto TiO₂ nanotube surfaces by electrostatic attraction. Moreover, the samples of assembled PLL or/and DNA had better bioactivity in inducing HA formation and promoting osteoblast cells adhesion, proliferation and early differentiation.     

Polyelectrolyte blend multilayer films: surface morphology, wettability, and protein adsorption characteristics

We report the influence of polyelectrolyte (PE) multilayer films prepared from poly(styrene sulfonate)-poly(acrylic acid) (PSS-PAA) blends, deposited in alternation with poly(allylamine hydrochloride) (PAH), on film wettability and the adsorption behavior of the protein immunoglobulin G (IgG). Variations in the chemical composition of the PAH/(PSS-PAA) multilayer films, controlled by the PSS/PAA blend ratio in the dipping solutions, were used to systematically control film thickness, surface morphology, surface wettability, and IgG adsorption. Spectroscopic ellipsometry measurements indicate that increasing the PSS content in the blend solutions results in a systematic decrease in film thickness. Increasing the PSS content in the blend solutions also leads to a reduction in film surface roughness (as measured by atomic force microscopy), with a corresponding increase in surface hydrophobicity. Advancing contact angles (theta) range from 7 degrees for PAH/PAA films through to 53 degrees for PAH/PSS films. X-ray photoelectron spectroscopy measurements indicate that the increase in film hydrophobicity is due to an increase in PSS concentration at the film surface. In addition, the influence of added electrolyte in the PE solutions was investigated. Adsorption from PE solutions containing added salt favors PSS adsorption and results in more hydrophobic films. The amount of IgG adsorbed on the multilayer films systematically increased on films assembled from blends with increasing PSS content, suggesting strong interactions between PSS in the multilayer films and IgG. Hence, multilayer films prepared from blended PE solutions can be used to tune film thickness and composition, as well as wetting and protein adsorption characteristics.     

Oligonucleotide fingerprinting of plant and fungal genomes: a comparison of radioactive, colorigenic and chemiluminescent detection methods.

Digoxigenated oligonucleotide probes complementary to simple repetitive DNA sequences were introduced into nonradioactive fingerprint analysis of plant and fungal DNA. The fragment patterns, obtained by blot hybridization of TaqI-restricted DNA from chickpea (Cicer arietinum) and its fungal pathogen Ascochyta rabiei with digoxigenated probes and either a colorigenic or a chemiluminescent detection method, were compared to those obtained with 32P-labeled probes. In combination with alkaline phosphatase and its chemiluminescent substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD) digoxigenated oligonucleotides yielded clear-cut fingerprints with high signal-to-background ratios within several minutes of exposure to X-ray films. The chemiluminescence reaction remained stable for at least two weeks. A comparison of banding patterns obtained by radioactive versus digoxigenin-based hybridization and detection techniques revealed substantial differences in the relative signal intensities of bands. Both nonradioactive techniques show a tendency to "equalize" band intensity differences. Whereas 32P-labeled oligonucleotides are also applicable to in situ hybridization with DNA immobilized in dried agarose gels, gel hybridization did not work efficiently with digoxigenated probes and either substrate.     

Direct observation of guanine radical cation deprotonation in duplex DNA using pulse radiolysis

The dynamics of one-electron oxidation of guanine (G) base mononucleotide and that in DNA have been investigated by pulse radiolysis. The radical cation (G+*) of deoxyguanosine (dG), produced by oxidation with SO(4)-*, rapidly deprotonates to form the neutral G radical (G(-H)*) with a rate constant of 1.8 x 10(7) s(-1) at pH 7.0, as judged from transient spectroscopy. With experiments using different double-stranded oligonucleotides containing G, GG, and GGG sequences, the absorbance increases at 625 nm, characteristic of formation of the G(-H)*, were found to consist of two phases. The rate constants of the faster ( approximately 1.3 x 10(7) s(-1)) and slower phases ( approximately 3.0 x 10(6) s(-1)) were similar for the different oligonucleotides. On the other hand, in the oligonucleotide containing G located at the 5'- and 3'-terminal positions, only the faster phase was seen. These results suggest that the lifetime of the radical cation of the G:C base pair (GC+*), depending on its location in the DNA chain, is longer than that of free dG. In addition, the absorption spectral intermediates showed that hole transport to a specific G site within a 12-13mer double-stranded oligonucleotide is complete within 50 ns; that is, the rate of hole transport over 20 A is >10(7) s(-1).     

层-层自组装构建固相可降解基因传递体系的研究

近年来,随着人类对基因研究的深入,基因治疗作为一种新的手段,受到人们的广泛重视．在组织工程材料、介入医用材料和医用植入体的应用中,与传统的溶液给药方式不同,基因技术需要一种可直接作用于材料表面贴壁细胞的长效、高转染固相基因传递体系．目前,国内外研究者将蛋白质药     

X-ray nanoscale profiling of layer-by-layer assembled metal/organophosphonate films

The nanoscale structure of multilayer metal/phosphonate thin films prepared via a layer-by-layer assembly process was studied using specular X-ray reflectivity (XRR), X-ray fluorescence (XRF), and long-period X-ray standing wave (XSW) analysis. After the SiO(2) X-ray mirror surfaces were functionalized with a monolayer film terminated with phosphonate groups, the organic multilayer films were assembled by alternating immersions in (a) aqueous solutions containing Zr(4+), Hf(4+), or Y(3+) cations and then (b) organic solvent solutions of PO(3)-R-PO(3), where R was a porphyrin or porphyrin-square spacer molecule. The different heavy metal cations provided X-ray fluorescence marker layers at different heights within the different multilayer assemblies. The XSW measurements used a 22 nm period Si/Mo multilayer mirror. The long-period XSW generated by the zeroth-order (total external reflection) through fourth-order Bragg diffraction conditions made it possible to examine the Fourier transforms of the fluorescent atom distributions over a much larger q(z)() range in reciprocal space than previously achieved.     

Dense passivating poly(ethylene glycol) films on indium tin oxide substrates

We describe the formation and characterization of surface-passivating poly(ethylene glycol) (PEG) films on indium tin oxide (ITO) glass substrates. PEG chains with a molecular weight of 2000 and 5000 D were covalently attached to the substrates in a systematic approach using different coupling schemes. The coupling strategies included the direct grafting with PEG-silane, PEG-methacrylate, and PEG-bis(amine), as well as the two-step functionalization with aldehyde-bearing silane films and subsequent coupling with PEG-bis(amine). Elemental analysis by X-ray photoelectron spectroscopy (XPS) confirmed the successful surface modification, and XPS and ellipsometry provided values for film thicknesses. XPS and ellipsometry thickness values were almost identical for PEG-silane films but differed by up to 400% for the other PEG layers, suggesting a homogeneous layer for PEG-silane but an inhomogeneous distribution for other PEG coatings on the molecularly rough ITO substrates. Atomic force microscopy (AFM) and water contact angle goniometry confirmed the different degrees of surface homogeneity of the polymer films, with PEG-silane reducing the AFM rms surface roughness by 50% and the water contact angle hysteresis by 75% compared to uncoated ITO. The ability of the PEG layers to passivate the substrate against the nonspecific adsorption of biopolymers was tested using fluorescence-labeled immunoglobulin G and DNA oligonucleotides in combination with fluorescence microscopy. The results indicate a positive relationship between film density and homogeneity on one hand and the ability to passivate against biopolymer adhesion on the other hand. The most homogeneous layers prepared with PEG-silane reduced the nonspecific adsorption of fluorescence-labeled DNA by a factor of 300 compared to uncoated ITO. In addition, the study finds that the ratio of film thicknesses derived by ellipsometry and XPS is a useful parameter to quantify the structural integrity of PEG layers on molecularly rough ITO surfaces. The findings may be applied to characterize PEG or other polymeric films on similarly coarse substrates.