DNA detection and cell adhesion on plasma‐polymerized pyrrole

This study investigates the application of Plasma‐polymerized pyrrole (ppPY) as bioactive platform for DNA immobilization and cell adhesion based on the fundamental properties of ppPY, such as chemical structure, electrochemical property, and protein adsorption. Variations in electrochemical properties of the ppPY film deposited under different plasma conditions before and after DNA immobilization were measured using electrochemical impedance spectroscopy (EIS). The equilibrium concentration of the probe DNA immobilized on the ppPY surface was deduced by detecting the variations in the surface charge transfer resistance (R_ct) of the ppPY films after DNA immobilization with different concentrations. In addition, the detection limit of the target DNA hybridization with probe DNA, the association constant, K_a, and the dissociation constant were deduced from Langmuir isotherm equations simulated using the experimental data collected by EIS. Moreover, inverted microscope was used to observe the cell adhesions onto the surface of the ppPY films prepared under different plasma conditions. Different adhesive behaviors of cells were observed, demonstrating that ppPY films could be an alternative biomaterial used as the sensitive layer for DNA sensor or cell adhesion. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 496–503, 2014.     

Protein immobilization on plasma-polymerized ethylenediamine-coated glass slides

For protein chip construction, protein immobilization on the surface of the glass slide is essential. It was previously reported that glass slides are embedded with chemicals that contain primary amines and aldehydes for protein immobilization. We fabricated a plasma-polymerized ethylenediamine (PPEDA)-coated slide that exposed primary amines. For the plasma polymer deposition on the glass slide, the inductively coupled plasma (ICP) power was found to be a critical factor in sustaining a high density of amine on the surface of the PPEDA films. We prepared PPEDA-coated slides at three different ICP powers (3, 30, or 70 W). In the slide that was prepared at a low ICP power (3 W), we detected a high density of primary amine. Therefore, the fluorescein isothiocyanate-conjugated immunoglobulin G (IgG) was highly immobilized to the PPEDA-coated slide that was prepared at the low ICP power. For protein immobilization, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) was used as a cross-linker. The immobilization of the protein to the PPEDA-coated slide was carried out by consecutive incubations with 1 mg/ml EDC for 5 min and 0.1 mg/ml IgG for 1 h. This efficiently produced the functionally active protein-immobilized slide. Therefore, this work shows that the plasma technique can be applied to produce a high-quality glass surface for the immobilization of proteins and other materials.     

The effect of the oxidation state of a terthiophene-conducting polymer and of the presence of a redox probe on its gene-sensing properties

The gene-sensing properties of sensor films made of a terthiophene-conducting polymer, poly(3-((2':2', 5':2'-terthiophene)-3'-yl)acrylic acid) (PTAA), were evaluated using electrochemical impedance spectroscopy for films in their reduced and oxidised states with and without the Fe(CN)(6)(3-/4-) redox probe (RP) in dilute tris-EDTA buffer. Porous films of PTAA were prepared and attached to an oligonucleotide sequence specific to the Salmonella virulence gene InvA. These films could be described with a dual transmission line model in which the polymer conductivity was increased as a consequence of surface binding of complementary DNA. The effect is analogous to that reported for silicon nanowires and field-effect transistors in dilute electrolyte modified by charge exchange across the polymer-electrolyte interface. As a result, gene sensing could be conveniently observed as a change in the impedance phase angle at a fixed frequency.     

Cold plasma functionalized TeraHertz BioMEMS for enzyme reaction analysis

In this paper, we describe the development, functionalization and functionality testing of a TeraHertz (THz) Bio-MicroElectroMechanical System (BioMEMS) dedicated to enzyme reaction analysis. The microdevice was fabricated by mixing clean room microfabrication with cold plasma deposition. The first is used to build the microfluidic circuits and the THz sensor, while the later serves for the polymerization of allylamine using a homemade glow discharge plasma reactor for a subsequent immobilization of enzymatic biocatalysts. Thermal stability of the deposited plasma polymer has been investigated by infrared spectroscopy. Fluorescent detection confirmed the efficiency of the immobilization and the enzyme hydrolysis into the BioMEMS microchannels. For the first time, the progression of the hydrolysis reaction over time was monitored by the THz sensor connected to a vectorial network analyzer. Preliminary results showed that sub-THz transmission measurements are able to discriminate different solid films, various aqueous media and exhibit specific transmission behavior for the enzyme hydrolysis reaction in the spectral range 0.06–0.11 THz.     

Reactive thin polymer films as platforms for the immobilization of biomolecules

Spin-coated thin films of poly(N-hydroxysuccinimidyl methacrylate) (PNHSMA) on oxidized silicon and gold surfaces were investigated as reactive layers for obtaining platforms for biomolecule immobilization with high molecular loading. The surface reactivity of PNHSMA films in coupling reactions with various primary amines, including amine-terminated poly(ethylene glycol) (PEG-NH2) and fluoresceinamine, was determined by Fourier transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), fluorescence microscopy, and ellipsometry measurements, respectively. The rate constants of PEG-NH2 attachment on the PNHSMA films were found to be significantly increased compared to the coupling on self-assembled monolayers (SAMs) of 11,11'-dithiobis(N-hydroxysuccinimidylundecanoate) (NHS-C10) on gold under the same conditions. More significantly, the PEG loading observed was about 3 times higher for the polymer thin films. These data indicate that the coupling reactions are not limited to the very surface of the polymer films, but proceed into the near-surface regions of the films. PNHSMA films were shown to be stable in contact with aqueous buffer; the swelling analysis, as performed by atomic force microscopy (AFM), indicated a film thickness independent swelling of approximately 2 nm. An increased loading was also observed by surface plasmon resonance for the covalent immobilization of amino-functionalized probe DNA. Hybridization of fluorescently labeled target DNA was successfully detected by fluorescence microscopy and surface plasmon resonance enhanced fluorescence spectroscopy (SPFS), thereby demonstrating that thin films of PNHSMA comprise an attractive and simple platform for the immobilization of biomolecules with high densities.     

One step processing of aminofunctionalized gate oxides

A plasma discharge process has been developed that allows the growth of biosensor gate oxides with adapted surface properties for the direct application of biomolecular immobilization cascades. The process involves an accurate selection of processing conditions, mainly, low temperature evaporation of (3-aminopropyl)triethoxysilane (APTS) and dynamic power and flow conditions. Room temperature evaporation of APTS was achieved by designing a vessel with an internal capillary network. The initial high power (100 W) plasma conditions were replaced by milder molecular fragmentation (50 W, 25 W) in a pure Ar discharge. Under these conditions the thin SiO₂ layers presented graded properties with a denser layer at the Si (100) interface and a hybrid organic–inorganic structure at the surface. The chemistry of the films was analysed by Fourier transformed infrared spectroscopy (FTIR) and Rutherford backscattering spectroscopy combined with elastic recoil detection analysis (RBS, ERDA), which confirmed the presence of the SiO₂ and organic phases. Contact angle measurements indicate the higher contribution of the basic polar component to the surface free energy. Furthermore, the higher affinity of the surface towards biomolecular immobilization was confirmed by fluorescence microscopy. Finally, penetration of nitrobenzaldehyde was obtained by application of a molecular permeation method evaluated by UV–vis spectroscopy onto fused silica substrates.     

Plasma polymerized epoxide functional surfaces for DNA probe immobilization

The development of functional surfaces for the immobilization of DNA probe is crucial for a successful design of a DNA sensor. In this report, epoxide functional thin films were achieved simply by pulsed plasma polymerization (PP) of glycidyl methacrylate (GMA) at low duty cycle. The presence of epoxide groups in the resulting ppGMA films was confirmed by Fourier transform infrared spectroscopy. The ppGMA coatings were found to be resistant to the non-specific adsorption of DNA strands, while the epoxide groups obtained could react with amine-modified DNA probes in a mild basic environment without any activation steps. A DNA sensor was made, and was successfully employed to distinguish different DNA sequences with one base pair mismatch as seen by surface plasmon enhanced fluorescence spectroscopy (SPFS). The regeneration of the present DNA sensor was also discussed. This result suggests that surface modification with ppGMA films is very promising for the fabrication of various DNA sensors.     

Electrochemical studies of DNA immobilization onto the azide-terminated monolayers and its interaction with taxol

A surface modification procedure for the creation of self-assembled monolayers (SAMs) that can be used as a scaffold for double-stranded DNA (dsDNA) incorporation onto the gold surfaces is described. The SAMs of an azidohexane thiol derivative were prepared on the Au electrode and then used for the immobilization of dsDNA. The electrochemical characteristics of dsDNA onto the SAM-modified gold electrode were investigated by cyclic voltammetry and electrochemical impedance spectroscopy, and the surface concentration of dsDNA onto the SAMs surface was estimated. The interaction of dsDNA with the anticancer drug, taxol (paclitaxel), was also studied on the surface of DNA/SAM/Au electrode. The observed decrease in the guanine oxidation peak current was used to monitor the interaction of taxol with DNA. The resulting Langmuir isotherm for taxol binding to DNA at the modified electrode was used to evaluate the binding constant of taxol-DNA. The results obtained supported the groove binding interaction of taxol with DNA. The modified electrode was used as a sensitive sensor for quantification of taxol in human serum sample.     

Modification of bacterial structures by a low‐temperature gas plasma and influence on packaging material

Aims: To investigate the effect of a cascaded dielectric barrier discharge (CDBD) treatment on the biological structure of a selected bacterium and on the properties of different polymer films. Methods and Results: Inactivation kinetics were measured using air as the process gas and using Bacillus atrophaeus spores and vegetative cells, which had been homogeneously distributed on a surface. The changes to the outer coats and the DNA of the endospores and cells after plasma treatment were determined using biomolecular and chemical methods. The experiments showed that damage to the DNA molecules and changes in the cell walls can be observed as a consequence of the CDBD treatment. Furthermore, the influence of the plasma treatment on the properties of various polymer films was investigated using a variety of test methods. Except the sealing strength where a slight decrease was observed (max. 20%), no negative changes of the material properties have occurred. Conclusions: CDBD treatment can affect the DNA of spores and cells, depending on the treatment time. At the same time, practically relevant inactivation rates on packaging materials were observed, without any significant changes to the material properties. Significance and Impact of the Study: Knowledge about CDBD mechanisms was acquired from a biological point of view, and the suitability of the method for treating polymer films was demonstrated.     

Electrochemical impedance detection of DNA hybridization based on dendrimer modified electrode

Bioactive ultrathin films with the incorporation of amino-terminated G4 PAMAM dendrimers have been prepared via layer-by-layer self-assembly methods on a gold electrode and used for the DNA hybridization analysis. Surface plasmon resonance (SPR), X-ray photoelectron spectroscopy (XPS), and electrochemical impedance spectroscopy (EIS) are used to characterize the successful construction of the multicomponent film on the gold substrate. The dendrimer-modified surfaces improve the immobilization capacity of the probe DNA greatly, compared to the AET (2-aminoethanethiol) SAM sensor surfaces without dendrimer molecules. DNA hybridization analysis is monitored by EIS. The dendrimer-based electrochemical impedance DNA biosensor shows high sensitivity and selectivity for DNA hybridization assay. The multicomponent films also display a high stability during repeated regeneration and hybridization cycles.     

An amperometric immunosensor for osteoproteogerin based on gold nanoparticles deposited conducting polymer

An amperometric immunosensor was fabricated for the detection of osteoproteogerin (OPG) by covalently immobilizing a monoclonal OPG antibody (anti-OPG) onto the gold nanoparticles (AuNPs) deposited functionalized conducting polymer (5,2′:5′,2″-terthiophene-3′-carboxylic acid). AuNPs were electrochemically deposited onto the conducting polymer using cyclic voltammetry. The particle size of deposited AuNPs was controlled by varying the scan rate and was characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The immobilization of anti-OPG was also confirmed using XPS. The principle of immunosensor was based on a competitive immunoassay between free-OPG and labeled-OPG for the active sites of anti-OPG. HRP was used as a label that electrochemically catalyzes the H₂O₂ reduction. The catalytic reduction was monitored amperometrically at −0.4 V vs. Ag/AgCl. The immunosensor showed a linear range between 2.5 and 25 pg/ml and the detection limit was determined to be 2 pg/ml. The proposed immunosensor was successfully applied for real human samples to detect OPG.     

Synthesis and characterization of biocompatible polymer interlayers on titanium implant materials

A bifunctional copolymer series of (4-vinylbenzyl)phosphonic acid diethylester and N-acryloxysuccinimide was developed as an interlayer with the aim of immobilizing proteins on titanium surfaces. Copolymers with varying compositions were synthesized, and an alternating copolymerization of the two monomers was found. The copolymers form ultrathin films of about 2-8 nm on titanium surfaces in a simple dipping process, as estimated from the attenuation of the titanium X-ray photoelectron spectroscopy (Ti-XPS) signal. The films were characterized by infrared spectroscopy, XPS, and time-of-flight secondary ion mass spectrometry. The results indicate that the immobilization is due to phosphonate groups, and thus the phosphonate content of the copolymers is decisive for the final film thickness. These polymer films were examined for their potential protein binding capacity by using trifluoroethylamine derivatization and subsequent XPS analysis as a reactivity assay.     

Environmental responsiveness of polygalacturonic acid-based multilayers to variation of pH

The effect of pH on the stability of layer-by-layer deposited polygalacturonic acid (PGalA)-based multilayer films prepared with the polycations poly-L-lysine, chitosan, and lysozyme is studied. The response was characterized using a quartz crystal microbalance, dual polarization interferometry, and Fourier transform infrared spectroscopy which probe multilayer thickness, density, polymer mass (composition and speciation), and hydration. All multilayers showed irreversible changes in response to pH change becoming thinner due to the partial disassembly. Preferential loss of the polycation (50-80% w/w) and relative small losses of PGaLA (10-35% w/w) occurred. The charge density on the polycation has a strong influence on the response to the acid cycle. Most of the disassembly takes place at the pH lower that pK(a) of PGaLA, indicating that this factor was crucial in determining the stability of the films. The pH challenge also revealed a polycation-dependent shift to acid pH in the PGaLA pK(a).     

Directed immobilization of nucleic acids at ultramicroelectrodes using a novel electro-deposited polymer

A two-step method for the directed immobilization of nucleic acids at ultramicroelectrodes with micron-size dimensions is described. The approach is based on the immobilization of streptavidin at the surface of carbon or noble metal electrodes within a novel electro-deposited polymer, formed by electropolymerization of the natural compound scopoletin (7-hydroxy-6-methoxy-coumarin) at potentials between 0.4 and 0.7 V vs. Ag/AgCl. Biotin-tagged nucleic acids or proteins are immobilized on top of the modified electrodes in a second step. The new method has some advantages compared to classical electropolymerization approaches (e.g. polypyrrole, polyphenol), because the growing polymer is highly hydrophilic, resulting in efficient incorporation of streptavidin and a high biotin binding capacity of 6 pmol cm(-2). The polymer film seems to be non-conductive but shows good swelling properties in aqueous solutions. The feasibility of the method for the electro-directed biochemical modification of individual microelectrodes has been demonstrated by sequential immobilization of two different single strand oligonucleotides onto interdigitated ultramicroelectrodes. The resulting miniature DNA probe was used for single base mutation detection with two synthetic targets (fluorescence-labeled 17-mer oligomers) by evaluating the fluorescence patterns after hybridisation with the immobilised DNA probes. The new method is useful for the production of microelectrode based DNA chips and for the electro-directed immobilisation of biomolecules at microelectrode structures with high spatial resolution and yield.     

Innovative metal oxide-based substrates for DNA microarrays

In the present report, we propose a novel approach to synthesize DNA microarrays that employs immobilization of the nucleic acid molecules onto zinc and iron oxide surfaces through their phosphate backbone. Oxide films were prepared by the sol–gel technique and the resulting surfaces were characterized especially with respect to surface chemistry and morphological features by both X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). ZnO films annealed at T ? 300 °C show the most promising surface features to be employed for DNA microarray preparation, i.e. high density of binding sites (hydroxyl groups), smooth and homogeneous surfaces, high optical transmittance in the visible spectral range suitable for detection using fluorescence, and easy handling during preparation procedures. The analysis of nucleic acid retention on the oxide layers was performed by the scanning of dye-labelled DNA previously printed on the substrate using the DNA microarray robotic arm. Clearly visible spots with regular shape were revealed above the background noise indicating that anchoring of the DNA on the treated surface is efficient and does not interfere with hybridization processes. The use of suitably engineered zinc oxide film makes the immobilization strategy ideal for facile, efficient, and cost-effective manufacturing of DNA microarrays.     

Fabrication of Large-scale Nanostructure by Langmuir-Blodgett Technique

Monolayer of polymer latex spheres was prepared at the air/water interface and deposited onto glass slides through Langmuir-Blodgett （LB） technique. Large-scale, high quality hexagonally close-packed domains were found in scanning electron microscopic pictures. Details of the monolayer-forming ability were discussed. Suitable surface characteristics of the colloidal particles, especially the hydrophilic and hydrophobic properties, are the keys for the formation of ordered monolayer films. The film can be transferred onto various kinds of substrates, even high curvature surface articles, such as fibers, decorations etc, can also be used as substrates. The advantages of this fabrication method of polymer latex spheres monolayer are fast, flexible, simple and very neat.     

Adsorption of dansylated amino acids on molecularly imprinted surfaces: a surface plasmon resonance study

Surface plasmon resonance spectroscopy (SPR) was used to measure the adsorption kinetics and isotherms of dansylated amino acids onto surface-confined molecularly imprinted polymer films (MIP-Fs) and the corresponding non-imprinted polymer control films (NIP-Fs). The surface-confined polymer films were grafted from flat gold surfaces using atom transfer radical polymerization (ATRP). This approach allowed uniform nanothin films to be grown, thereby ensuring that the amino acids see a uniform surface during adsorption. N,N'-Didansyl-l-cystine (DDC) and didansyl-l-lysine (DDK) were used as the template molecules to form the MIP-Fs. Adsorption kinetics data were analyzed using single- and dual-site Langmuir adsorption models. It was found that, within the experimental measurement range, adsorption isotherm data were well described by any of four isotherm models: Langmuir, dual-site Langmuir, Freundlich, or Langmuir-Freundlich (LF). The relatively high heterogeneity index values regressed using the Freundlich and LF isotherms suggest the formation of fairly homogeneous MIP-Fs; although Scatchard analysis reveals binding site heterogeneity does exist. Selectivity studies showed that the MIP-Fs display cross-reactivity between DDC and DDK; nevertheless, MIP-Fs prepared against one template showed selectivity for that template. Solution pH and polymer layer thickness were studied as independent parameters to determine their impacts on amino acid adsorption, as monitored by SPR.     

Simultaneous purification and immobilization of Aspergillus niger xylanase on the reversibly soluble polymer Eudragit(TM) L-100

The non-covalent immobilization of a commercial preparation of xylanase from A. niger was carried out on a reversibly soluble-insoluble enteric polymer Eudragit(TM) L-100. The immobilization of the xylanase activity by adsorption was simultaneously accompanied by removal of cellulase activity since the latter did not bind to the polymer. Thus, the soluble enzyme derivative may be useful for treatment of paper pulp bleaching in paper industry. The immobilized xylanase retained 60% of its activity toward xylan as the substrate. No change was observed in the pH optimum (5.5) of the enzyme upon immobilization. Only marginal increase in the K(m) of the free enzyme (3.6 mg ml(-1) to 5.0 mg ml(-1)) upon immobilization on the soluble polymer reflected that the enzyme-substrate binding continues to be efficient in spite of the macromolecular nature of the substrate. Fluorescence spectroscopy and UV difference spectroscopy were used to probe the change(s) in the enzyme structure upon immobilization. This change in structure was correlated with the "effectiveness factor" of the enzyme activity. CD spectra also showed that the enzyme undergoes drastic changes in the structure.     

The investigation of protein adsorption behaviors on different functionalized polymers films

The adsorption of BSA and fibrinogen onto plasma-polymerized di-(ethylene glycol) vinyl ether, allylamine, and maleic anhydride films were investigated in detail by surface plasmon resonance spectroscopy (SPR). The chemical properties of the plasma polymers were initially determined by the plasma deposition conditions during the generation procedure. The analysis of the chemical structure of the films and the refractive index of plasma polymers in aqueous solution was carried out using Fourier transform infrared spectroscopy and waveguide mode spectroscopy, respectively. Using water contact angle measurement, the surface wettability of plasma polymers was also characterized. These properties have a critical influence on the behavior of protein adsorption on the surface of the plasma polymers. Protein adsorption was found to depend not only on the types of functionalized groups, but also on the plasma polymer thickness since the protein molecules penetrate into the plasma polymer network bulk. According to the size of protein molecules in aqueous solution and the amount of adsorbed proteins observed by SPR, the conformational changes of proteins could be deduced.     

Enzymatic activities and infrared studies of glutamate dehydrogenase immobilized on Langmuir-Blodgett films

Summary A technique is described for the immobilization of active glutamate dehydrogenase (GDH) on behenic acid Langmuir-Blodgett (LB) films. The optimization of the immobilization conditions shows that the activities of GDH bound on hydrophobic and hydrophilic LB films were similar and decreased dramatically when the immobilized enzyme was dried. The GDH binding was followed by Fourier transform infrared (FTIR) spectroscopy. Modifications of GDH conformation and LB film structure were observed during the enzyme binding. After GDH activity test, a partial dissociation of behenic acid occurred and the -sheet band of the enzyme increased by comparison with the -helix band.Abbreviations LB Langmuir-Blodgett - FTIR spectroscopy Fourier transform infrared spectroscopy - GDH glutamate dehydrogenase - TEA triethylamine