Thermal-sensitive PBMA-b-PNIPAAm amphiphilic block copolymers brushes and their assembling micelles

Brush-like block copolymers with poly(t-butyl methacrylate) (PBMA) and poly(N-ispropylacrylamide) (PNIPAAm) as side arms, PBMA-b-PNIPAAm, were designed and synthesized via a simple free radical polymerization route. The chemical structure of these polymer brushes was characterized and determined by nuclear magnetic resonance (1H NMR), and Fourier transform infrared spectrometry (FT-IR). The micellar formation by these polymer brushes in aqueous solutions were detected by a surface tension technique, and the critical micelle concentration (CMC) ranged from 1.53 to 8.06 mg L(-1). The morphology and geometry of polymer micelles were investigated by transmission electron microscope (TEM) and dynamic light scattering (DLS). The polymer micelles assume the regularly-spherical core-shell structure with well-dispersed individual nanoparticles, and the particle size was in the range from 36 to 93 nm. The PNIPAAm segments exhibited a thermoreversible phase transition, so the resulting block polymer brushes were temperature-sensitive and the low critical solution temperature (LCST) was determined by UV-vis spectrometer at about 28.82-29.40 degrees C. The characteristic parameters of the polymer micelles such as CMC, micellar size and LCST values were affected by their compositional ratios and the length of hydrophilic or hydrophobic chains. The self-assembled micelles are expected to be used in specific biomedical fields as a candidate of drug controlled release carrier.     

Characterization of a polymeric adsorbed coating for DNA microarray glass slides

A new method was developed to covalently attach target molecules onto the surface of glass substrates such as microwell plates, beads, tubes, and microscope slides, for hybridization assays with fluorescent targets. The innovative concept introduced by this work is to physically adsorb onto underivatized glass surfaces a functional copolymer, able to graft amino-modified DNA molecules. The polymer, obtained by radical copolymerization of N,N-dimethylacrylamide, N-acryloyloxysuccinimide, and 3-(trimethoxysilyl)propyl methacrylate, copoly(DMA-NAS-MAPS), self-adsorbs onto the glass surface very quickly, typically in 5-30 min. The film, formed on the surface, bears active esters, which react with amino-modified DNA targets. The surface layer is stable in an aqueous buffer containing various additives (SDS, urea, salt), even at boiling temperature. It should be emphasized that the coating is formed by the immersion of glass slides in a diluted aqueous solution of the polymer. Therefore, the procedure is fast, inexpensive, robust, and reliable, and it does not require time-consuming glass pretreatments. Slides, coated with copoly(DMA-NAS-MAPS), were profitably used as substrates for the preparation of low-density DNA microarrays. The density and the thickness of the films were evaluated by X-ray reflectivity measurements whereas the extent of reaction of functional groups with DNA molecules was determined by a functional test. The experiments indicate that half of the active groups present on the surface reacts with oligonucleotide probes.     

Nanomechanical Properties of Oligo(ethylene glycol methacrylate) Polymer Brush‐Based Biointerfaces

Protein resistant polymer brushes of poly(diethylene glycol methylether methacrylate) (DEGMA) are synthesized by surface‐initiated polymerization (SIP) on gold and are thoroughly characterized with particular attention to the nanomechanical properties. Compared to the well known brushes of poly(oligoethylene glycol methylether methacrylate) (POEGMA) on gold, PDEGMA brushes grow faster and yield higher final brush heights. In confocal fluorescence microscopy experiments it is shown that both types of brushes possess similar low non‐specific adsorption levels of labeled bovine serum albumin (BSA) from buffer. While the surface stiffness evaluated by depth sensing indentation in the dry state suggests that side chain crystallization imparts high stiffness to the POEGMA brush, PDEGMA brushes behave similar to poly(methyl methacrylate) (PMMA) brushes of comparable thickness. In the wet state PDEGMA brushes are shown to possess an elastic modulus of 800 kPa, as estimated by AFM nanoindentation, which is smaller than that of POEGMA (3 000 kPa). Expanding these brushes to end‐functional polymers with recently developed block copolymer brush approaches, tailored soft biointerfaces can be generated for cell ‐ surface interaction and cell culture studies.     

RAFT-mediated synthesis of cationic poly[(ar-vinylbenzyl)trimethylammonium chloride] brushes for quantitative DNA immobilization

The synthesis of cationic poly[(ar-vinylbenzyl)trimethylammonium chloride)] [poly(VBTAC)] brushes was achieved via reversible addition-fragmentation chain transfer (RAFT) polymerization and used for quantitative DNA immobilization. Initially, silicon surfaces were modified with RAFT chain transfer agent by utilizing an amide reaction involving a silicon wafer modified with allylamine and 4-cyanopentanoic acid dithiobenzoate (CPAD). Poly(VBTAC) brushes were then prepared via RAFT-mediated polymerization from the surface immobilized CPAD. Various characterization techniques including ellipsometry, X-ray photoelectron spectroscopy, grazing angle-Fourier transform infrared spectroscopy, atomic force microscopy and contact-angle goniometer were used to characterize the immobilization of CPAD on the silicon wafer and the subsequent polymer formation. The addition of free CPAD was required for the formation of well-defined polymer brushes, which subsequently resulted in the presence of free polymer chains in solution. The free polymer chains were isolated and used to estimate the molecular weights and polydispersity index of chains attached to the surface. Moreover, from atomic force microscopy and ellipsometry measurements, it was also determined that the density of immobilized DNA on the cationic poly(VBTAC) brushes can be quantitatively controlled by adjusting the solution concentration.     

Identification of p53 gene by using CdSe/ZnS conjugation and hybridization

Recently Quantum Dots (QDs) have been of great interest due to their unique optical properties such as size-dependent, symmetric, narrow, and stable emissions, allowing for prolonged observation and multiplexing. We have prepared oligonucleotide conjugated to QD as a probe to detect p53 tumor suppressor gene related to hereditary cancer. QDs with carboxyl functional group have been conjugated to thiol-modified oligo nucleotides, which have been used as a hybridization probe for p53 gene. Target gene was added and hybridized with the QD bound probe. The conjugation of QD and thiolated oligonucleotide was stained by gel electrophoresis using Etrium Bromide (EtBr) as intercalating dye. Fluorescence resonance energy transfer (FRET) has been observed between QD and intercalating dye (Propidium Iodide) after hybridization of target and probe. FRET efficiency was increased with the increase of dye and DNA concentration. This shows the possibility of specific detection of low concentration of unlabeled complimentary DNA via quantum dots.     

Label-free detection of DNA hybridization at a liquid|liquid interface

A novel electrochemical approach for label-free detection of DNA primary sequence has been proposed. The flow of nonelectroactive ions across a liquid|liquid interface was used as an electrochemical probe for detection of DNA hybridization. Disposable graphite screen-printed electrodes shielded with a thin layer of inert polymer plasticized with water-immiscible polar organic solvent were modified by probe oligonucleotide and used as a DNA sensor. The specific DNA coupling has been detected with impedance spectroscopy by decrease of ion-transfer resistance. The detection limit was of 10-8 M of target oligonucleotide. The reported sensor was suitable for discrimination of a single mismatch oligonucleotide from the full complementary one. The reported DNA sensor was advantageous over known physicochemical approaches, providing the most significant changes in the measured parameters.     

Probing the collapse dynamics of poly(N-isopropylacrylamide) brushes by AFM: effects of co-nonsolvency and grafting densities

Collapse of poly(N-isopropylacrylamide) (PNIPAM) brushes in the mixed solvent system (water/methanol 50% v/v) is studied by in-situ atomic-force microscopy (AFM). PNIPAM brushes with three different grafting densities and similar chain lengths are synthesized via surface-initiated atom-transfer radical polymerization. By changing the solvent from water to a water/methanol (50% v/v) mixture, the polymer brushes switch from a swollen to collapsed state. AFM force measurements using a silica colloidal probe attached to the tip are employed to obtain the Young's moduli of the polymer brushes in different solvation states. The collapse dynamics of the brush is followed by monitoring the pull-off force (adherence) in situ. The modulus of the swollen high-density polymer brush is four times lower than that of the same brush in the collapsed state. It is shown that in the case of the high-density polymer brush with a thickness (t(in water) ) of 900 nm, the collapse takes place in a time scale of ~25 s, whereas the collapse occurs faster for the medium-density brush (t(in water) = 630 nm) and much more rapidly for the low-density brush (t(in water) = 80 nm). This difference in the response kinetics is primarily ascribed to the time needed for solvent exchange in the polymer brushes.     

Development of oligonucleotide microarray involving plasma polymerized acrylic acid

This paper presents the manufacturing of biochips by using the COOH- derived polymer coating deposited by plasma polymerization of acrylic acid. This technology is based on depositing a thin layer obtained by plasma polymerization of acrylic acid which allows a further covalent immobilization of biomolecules on glass substrates. The plasma power value was optimized to maximize the stability of plasma polymerized acrylic acid (PPAA) coatings in water, which has a very important role for such applications. In order to obtain a covalent immobilization of DNA probes on the PPAA coated surface, the activation protocol of carboxylic function was carried out with the help of N-Hydroxy Succinimide and 1-Ethyl-3-(3-DimethylAminopropyl) Carbodiimide. The efficiency of PPAA coated in microarray applications was compared with two types of commercial slides. Such surfaces have shown very interesting results in terms of relative density of attached DNA probe molecules and signal-to-background ratio measured for target DNA hybridization. Nonspecific DNA bonding measurements showed only a small amount of nonspecific physisorption between the DNA probe and the PPAA-activated surfaces. This work shows that the plasma polymerization technique can be successfully applied to produce a high-quality glass surface for the manufacturing of DNA arrays.     

Carbazole functionalized isocyanide brushes in heterojunction photovoltaic devices

In this work, carbazole-containing polyisocyanide (PIACz) brushes were used for photovoltaic devices. A photovoltaic device was fabricated on top of the brushes by spin-coating a suitable acceptor and evaporating an Al cathode. Devices with a poly(N-vinylcarbazole) (PVK) bulk polymer were also prepared for comparison. Interestingly, the brushes showed better photovoltaic characteristics as compared to the blended PVK system. This is attributed to the specific morphologies of the polyisocyanide brushes, which provide a large interfacial area between the donor and acceptor for efficient photogeneration. It was found that the device performance varied according to the molecular size of the incorporated acceptors.     

电化学诱导表面引发原子转移自由基聚合构筑离子型聚醚砜膜功能表面

采用电化学诱导表面引发原子转移自由基聚合(SI-eATRP)技术,在涂覆聚多巴胺的聚醚砜膜基底上接枝离子型聚合物分子刷聚对苯乙烯磺酸钠,并通过单体浓度对聚合物分子刷进行调控。采用SEM、AFM、XPS、水接触角等表征方法对改性聚醚砜膜的结构和性能进行表征;采用水通量对其进行滤过性能测定。结果表明:成功地在改性聚醚砜膜表面接枝离子型聚合物分子刷聚对苯乙烯磺酸钠;聚合物刷相互缠结形成了球状颗粒;随着电化学诱导体系中单体浓度的增大,聚合物分子刷的接枝量增加,同时水接触角显著降低;聚合物膜表面离子型分子刷的构筑改善了亲水性,因此其纯水通量明显增加,牛血清白蛋白(BSA)截留率和通量恢复率都得到了提高。SI-eATRP用于聚合物膜材料的表面改性和调控,在生物相容性膜等领域具有潜在的应用前景。     

Direct DNA hybridization detection based on the oligonucleotide-functionalized conductive polymer

Electrochemical methods for DNA hybridization detection have many advantages that are very fast to detect hybridization and can be directly applied for a portable DNA sensor. In this paper, an electrochemical method to directly detect DNA hybridization was developed on the basis of a new conductive polymer, which was polymerized on the glassy carbon electrode with a terthiophene monomer having a carboxyl group (3'-carboxyl-5,2',5',2"-terthiophene). The ss-DNA probe was made by chemically bonding an amine-linked C6 alkyl group to the 5' terminus of oligonucleotide (19-mer). The probe moiety was immobilized on the polymer through covalent bonding with a catalyst, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. A difference in admittance was observed before and after hybridization as a result of the reduction of the resistance after hybridization. The highest difference in admittance was observed around 1 kHz before and after hybridization. Hybridization amounts of end two-base and center one-base mismatched sequences were obtained only in a 14.3% response when compared to that for the complementary matched sequence.     

Polymer brushes on carbon nanotubes by thiol-lactam initiated radical polymerization of 2-hydroxyethyl methacrylate

Water-soluble polymer brushes with multi-walled carbon nanotubes (MWNTs) as backbones were synthesized by grafting 2-hydroxyethyl methacrylate (HEMA) from surface functionalized MWNTs via in situ surface thiol-lactam initiated radical polymerization. MWNTs were functionalized with 2-mercaptoethanol and used as initiators in the polymerization of HEMA in the presence of butyrolactam. FT-IR, XPS, 1H NMR, GPC and TGA were used to determine chemical structure and the grafted polymer quantities of the resulting product. The covalent bonding of PHEMA to the MWNTs dramatically improved the water dispersibility of MWNTs. The average thicknesses of the polymer brushes in the functionalized MWNTs were detected with electron microscopy (SEM and TEM) and images indicated that the nanotubes were coated with polymer layer.     

Nanoparticle probes and mid-infrared chemical imaging for DNA microarray detection

To date most mid-infrared spectroscopic studies have been limited, due to lack of sensitivity, to the structural characterization of a single oligonucleotide probe immobilized over the entire surface of a gold-coated slide or other infrared substrate. By contrast, widely used and commercially available glass slides and a microarray spotter that prints approximately 120-μm-diameter DNA spots were employed in the present work. To our knowledge, mid-infrared chemical imaging (IRCI) in the external reflection mode has been applied in the present study for the first time to the detection of nanostructure-based DNA microarrays spotted on glass slides. Alkyl amine-modified oligonucleotide probes were immobilized on glass slides that had been prefunctionalized with succinimidyl ester groups. This molecular fluorophore-free method entailed the binding of gold-nanoparticle-streptavidin conjugates to biotinylated DNA targets. Hybridization was visualized by the silver enhancement of gold nanoparticles. The adlayer of silver, selectively bound only to hybridized spots in a microarray, formed the external reflective infrared substrate that was necessary for the detection of DNA hybridization by IRCI in the present proof-of-concept study. IRCI made it possible to discriminate between diffuse and specular external reflection modes. The promising qualitative results are presented herein, and the implications for quantitative determination of DNA microarrays are discussed.     

Detection of DNA point mutation by atom transfer radical polymerization

We report here a new DNA detection method in which polymer growth in atom transfer radical polymerization (ATRP) is used as a means to amplify detection signals. In this method, DNA hybridization and ligation reactions led to the attachment of ATRP initiators on a solid surface where specific DNA sequences were located. These initiators subsequently triggered the growth of poly(hydroxyethyl methacrylate) (PHEMA) at the end of immobilized DNA molecules and formed polymer brushes. The formation of PHEMA altered substrate opacity, rendering the corresponding spots readily distinguishable to the naked eye. A second ATRP reaction to form branched polymers on the surface drastically improved the visibility of DNA hybridization and significantly shortened the detection time. The resulting polymer film was characterized using infrared spectroscopy, ellipsometry, contact angle measurements, and atomic force microscopy. Direct visualization of 1 fmol of target DNA molecules of interest was demonstrated. A proof-of-principle experiment to detect DNA point mutation was conducted. The perfectly matched DNA targets were distinctively differentiated from those with mutations. The demonstrated capability to detect DNA mutation with direct visualization laid the groundwork for the future development of detector-free testing kits in single-nucleotide polymorphism screenings.     

Polymer brushes as active nanolayers for tunable bacteria adhesion

Bacterial biofilm formation on implant surfaces is a frequent reason for the failure of many biomedical devices. Polymer brushes, thin nanolayers constituted of densely grafted macromolecules, are promising candidates to use in many biomedical applications to control attachment of bacteria to a surface. In this work five different polymer brushes were synthesized and tested with respect to their ability to regulate Staphylococcus aureus adhesion. Namely, two mixed brushes [consisting of poly(ethylene glycol) and a positively charged polymers, poly(2-vynil pyridine) or quartenized poly(2-vynil pyridine)] are investigated along with one-component brushes of the respective polymers. Bacterial adhesion was regulated over two orders of magnitude via altering the polymer brush composition.     

A microfluidic platform using molecular beacon-based temperature calibration for thermal dehybridization of surface-bound DNA

This work presents a simple microfluidic device with an integrated thin-film heater for studies of DNA hybridization kinetics and double-stranded DNA melting temperature measurements. The heating characteristics of the device were evaluated with a novel, noninvasive indirect technique using molecular beacons as temperature probes inside reaction chambers. This is the first microfluidic device in which thermal dehybridization of surface-bound oligonucleotides was performed for measurement of double-stranded DNA melting temperatures with +/- 1 degrees C precision. Surface modification and oligonucleotide immobilization were performed by continuously flowing reagents through the microchannels. The resulting reproducibility of oligonucleotide surface densities, at 9% RSD, was better than for the same modification chemistries on glass slides in unstirred reagent solutions (RSD=20%). Moreover, the surface density of immobilized DNA probe molecules could be varied controllably by changing the concentration of the reagent solution used for immobilization. Thus, excellent control of surface characteristics was made possible, something which is often difficult to achieve with larger devices. Solid-phase hybridization reactions, a fundamental aspect of microarray technologies often taking several hours in conventional systems, were reduced to minutes in this device. It was also possible to determine forward rate constants for hybridization, k. These varied from 820,000 to 72,000 M(-1) s(-1), decreasing as surface densities increased. Surface densities could therefore be optimized to obtain rapid hybridization using such an approach. Taken together, this combined microfluidic/small-volume heating approach represents a powerful tool for surface-based DNA analysis.     

Patterned immobilization of biomolecules on a polymer surface functionalized by radiation grafting

Patterned graft polymerization of a functional monomer on a hydrophobic polymer surface was proposed for biomolecule patterning. A poly(vinylidene fluoride) (PVDF) film surface was selectively activated by ion implantation through a pattern mask and acrylic acid (AA) was then graft polymerized onto the activated regions of the PVDF surfaces. The peroxide concentration on the implanted surface depended on the fluence, which had a considerable effect on the grafting degree of AA. Afterwards, amine-functionalized biotin and probe DNA were immobilized on the poly(acrylic acid)-grafted regions of the PVDF surfaces. Specific binding of biotin with streptavidin and hybridization of probe DNA with complimentary DNA proved successful protein and DNA patterning and well-defined 50 microm dot-type patterns of the streptavidin and DNA were obtained. These results confirmed the potential of this strategy for patterning of various biomolecules.     

Electrochemical DNA sensors based on enzyme dendritic architectures: an approach for enhanced sensitivity

The modification of enzymes with multiple single-stranded oligonucleotides opens up a new concept for the development of DNA sensors with enhanced sensitivity. This work describes the generation of reporter sequences labeled with an enzyme for the demonstration of their ability to specifically hybridize and to permit signal amplification by successive hybridization steps. The synthetic pathway for the labeling of GOx with oligonucleotide sequences is based on the oxidation of the glycosidic residues of the enzyme and their covalent binding with 5'-end amine-modified oligonucleotides. Spectrophotometric characterization of these functionalized sequences results in an average number of three linked oligonucleotides per enzyme molecule. Their specificity is demonstrated in both a direct and a sandwich-type hybridization assay. The transduction of the enzyme-linked DNA sensors is based on self-assembled multilayers, including a chemically modified anionic horseradish peroxidase electrochemically connected to a water-soluble cationic poly[(vinylpyridine)Os(bpy)(2)Cl] redox polymer in an electrostatic ordered assembly. The sensing layer is constructed by the covalent binding of the DNA probe over the redox polymer through the 3'-phosphate group, enabling the capture of the target sequence. Upon addition of glucose, hybridization results in the production of H(2)O(2), which readily diffuses to the electrocatalytic assembly, giving rise to a cathodic current at 100 mV vs Ag/AgCl. Hybridization is always performed at room temperature, and after 30 min of incubation, an amperometric response is obtained that is proportional to DNA concentration. The simultaneous sandwich assay enables the quantification of a free-label 44-mer oligonucleotide at 1 nM concentration. Signal amplification is realized by a new hybridization step over the free sequences, giving rise to a dendritic architecture that accumulates enzyme molecules per hybridization event.     

Probing the responsive behavior of polyelectrolyte brushes using electrochemical impedance spectroscopy

Cyclic voltammetry and impedance spectroscopy were employed to probe the responsive properties of polyelectrolyte brushes. Poly[(dimethylamino)ethyl methacrylate] (PDMAEMA) brushes over 100 nm thick on gold substrates were synthesized via surface-initiated atom-transfer radical polymerization and quaternized with methane iodide to obtain cationic brushes (Q-PDMAEMA). Q-PDMAEMA brushes respond to electrolytes by exhibiting swollen and collapsed states. Swollen brushes allow good permeability of electroactive probes, while collapsed states block electron transport. Electrolytes have different impacts on the electrochemical properties of Q-PDMAEMA. Some salts (NaNO3) cause brush collapse due to charge screening, while others such as those with more hydrophobic anions (ClO4-, PF6-, and Tf2N-) induce brush collapse because of solubility changes. The collapsed brushes exhibit intrinsically different resistance as probed with impedance. Charged screened brushes retain good permeability to electroactive probes. Strongly coordinating hydrophobic anions lead to insoluble brushes, resulting in a high resistance. These results show that electrochemical impedance spectroscopy is a powerful technique to probe the properties and structure of polyelectrolyte brushes.     

PMMA brush-containing two-solution bone cement: preparation, characterization, and influence of composition on cement properties

Two-solution bone cement consisting of poly (methyl methacrylate) (PMMA) brushes in methyl methacrylate has been developed as an alternative to the traditional two-solution (TSBC) and powder-liquid cements. It was hypothesized that the substitution of brushes, for the entire pre-polymer phase of the cement, would permit a decrease in solution viscosity at higher polymer fractions, and allow for physical entanglements with the cement matrix. Consequently, improved cement exothermal and mechanical properties could be expected with brush addition. PMMA brushes were grafted on the surface of cross-linked PMMA nanospheres following a multi-stage synthetic strategy. Brushes exhibiting optimal molecular weight for preparation of TSBC were used for characterization of cement viscosity, flexural and compressive mechanical properties, exothermal properties and residual monomer content. Interactions between grafts and free polymer formed during free radical polymerization of the cement were evaluated based on molecular weight measurements of the cement matrix and brushes. Brush-containing cements exhibited lower viscosity at significantly higher polymer fractions in comparison to TSBC. Cements with PMMA brushes had significantly lower polymerization temperatures and residual monomer content. Measurements of molecular weight revealed the existence of a dry brush regime when using the brush compositions selected in this study, which led to a reduction in the mechanical properties of some of the compositions tested. The optimal cement viscosity and maintenance of other important cement properties achieved with addition of PMMA brushes is expected to expand the use of the two-solution cements in a range of applications.