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.     

DNA binding and alkylation by the "left half" of azinomycin B

Azinomycin B (also known as carzinophilin A) contains two electrophilic functional groups-an epoxide and an aziridine residue-that react with nucleophilic sites in duplex DNA to form cross-links at 5'-dGNT and 5'-dGNC sequences. Although the aziridine residue of azinomycin is undoubtedly required for cross-link formation, analogues containing an intact epoxide group but no aziridine residue retain significant biological activity. Azinomycin epoxide analogues (e.g., 5 and 6) are of interest due to their potent biological activity and because there is evidence that azinomycin may decompose in vivo to yield such compounds. To investigate the chemical events underlying the toxicity of azinomycin epoxides, DNA binding and alkylation by synthetic analogues of azinomycin B (6, 8, and 9) that comprise the naphthalene-containing "left half" of the antibiotic have been investigated. The epoxide-containing analogue of azinomycin (6) efficiently alkylates guanosine residues in duplex DNA. DNA alkylation by 6 is facilitated by noncovalent binding of the compound to the double helix. The results of UV-vis absorbance, fluorescence spectroscopy, DNA winding, viscometry, and equilibrium dialysis experiments indicate that the naphthalene group of azinomycin binds to DNA via intercalation. Equilibrium dialysis experiments provide an estimated binding constant of (1.3 +/- 0.3) x 10(3) M(-)(1) for the association of a nonalkylating azinomycin analogue (9) with duplex DNA. The DNA-binding and alkylating properties of the azinomycin epoxide 6 provide a basis for understanding the cytotoxicity of azinomycin analogues which contain an epoxide residue but no aziridine group and may provide insight into the mechanisms by which azinomycin forms interstrand DNA cross-links.     

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.     

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.     

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.     

A urea electrochemical sensor based on molecularly imprinted chitosan film doping with CdS quantum dots

An improved imprinted film-based electrochemical sensor for urea recognition was developed using CdS quantum dots (QDs) doped chitosan as the functional matrix. The microstructure and composition of the imprinted films depicted by scanning electron microscopy (SEM), attenuated total reflection infrared (ATR-IR), X-ray diffraction (XRD), and electrochemical impedance spectroscopy (EIS) indicated the fabricated feasibility of the nanoparticle doped films via in situ electrodeposition. Differential pulse voltammetric responses under the optimal fabrication conditions showed that the sensitivity of CdS QDs-MIP (molecularly imprinted polymer) electrochemical sensor was enhanced from the favorable electron transfer and magnified surface area of CdS QDs with a short adsorption equilibrium time (7 min), wide linear range (5.0 × 10(-12) to 4.0 × 10(-10) M and 5.0 × 10(-10) to 7.0 × 10(-8) M), and low detection limit (1.0 × 10(-12) M). Meanwhile, the fabricated sensor showed excellent specific recognition to template molecule among the structural similarities and coexistence substances. Furthermore, the proposed sensor was applied to determine the urea in human blood serum samples based on its good reproducibility and stability, and the acceptable recovery implied its feasibility for practical application.     

Gold Nanoring Arrays for Near Infrared Plasmonic Biosensing

Gold nanoring array surfaces that exhibit strong localized surface plasmon resonances (LSPR) at near infrared (NIR) wavelengths from 1.1 to 1.6 μm were used as highly sensitive real-time refractive index biosensors. Arrays of gold nanorings with tunable diameter, width, and spacing were created by the nanoscale electrodeposition of gold nanorings onto lithographically patterned nanohole array conductive surfaces over large areas (square centimeters). The bulk refractive index sensitivity of the gold nanoring arrays was determined to be up to 3,780 cm⁻¹/refractive index unit by monitoring shifts in the LSPR peak by FT-NIR transmittance spectroscopy measurements. As a first application, the surface polymerization reaction of dopamine to form polydopamine thin films on the nanoring sensor surface from aqueous solution was monitored with the real-time LSPR peak shift measurements. To demonstrate the utility of the gold nanoring arrays for LSPR biosensing, the hybridization adsorption of DNA-functionalized gold nanoparticles onto complementary DNA-functionalized gold nanoring arrays was monitored. The adsorption of DNA-modified gold nanoparticles onto nanoring arrays modified with mixed DNA monolayers that contained only 0.5 % complementary DNA was also detected; this relative surface coverage corresponds to the detection of DNA by hybridization adsorption from a 50 pM solution.

     

Biocompatibility and Conductometric Property of Sol-Gel Derived ZnO/PVP Nanocomposite Biosensor Film

Nanocrystalline ZnO and ZnO/PVP nanocomposite films have been prepared by the sol-gel dip-coating technique from zinc acetate precursor on silicon wafer and Pyrex glass substrates. The films were characterized using atomic force microscopy for morphology, and X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy for chemical analysis. Thermally untreated and annealed films were studied in order to analyze the influence of temperature on the formation and properties of the films. The films have a uniform void-free surface and the grain size increases with the annealing temperature. The cell viability assays indicate that the growth rate of BPH cells incubated in the presence of ZnO was significantly reduced (35% of the control) compared to that of untreated controls, indicating antibacterial activity of ZnO as a result of the generation of hydrogen peroxide. The sensor characteristic of ZnO/PVP nanocomposite was also demonstrated by measuring the change in conductivity upon exposure to superoxide anion radical.     

Fabrication of layer-by-layer deposited multilayer films containing DNA and its interaction with methyl green.

Multilayer films were fabricated by layer-by-layer electrostatic deposition techniques between poly(diallyldimethylammonium chloride) (PDDA) and calf thymus DNA (CT DNA) on glassy carbon and quartz substrates. Electrochemical impedance spectroscopy (EIS), Fourier transform infrared (FTIR) spectroscopy and UV-vis spectroscopy demonstrated the uniform assembly of PDDA/DNA multilayer films, and X-ray photoelectron spectroscopy confirmed the elemental composition of the films. Moreover, the interaction of DNA in PDDA/DNA films with methyl green was investigated by UV-vis spectroscopy and circular dichroism (CD).     

A disposable polymer sensor chip combined with micro-fluidics and surface plasmon read-out

This paper describes a new type of disposable polymeric sensor chip based on the grating coupling of surface plasmon modes combined with a micro-fluidic channel system. A specifically designed silicon stamp with nano-structure (grating) on the micro-structures (micro-channel) was fabricated by combining a holographic method and photolithography. By using such a stamp the micro-channels, the grating coupler and the gold which was first thermally evaporated onto the stamp were transferred to the polymeric substrate successfully in one step. It is demonstrated that the grating profile in the micro-channels allowed a very efficient coupling of the laser light to the surface plasmons propagating at the bottom of the micro-channels. The transferred gold exhibits properties of a freshly cleaned surface, and the self-assembly of a functional thiol derivative (mercapto-PEG) onto the sensor chip can be monitored by surface plasmon spectroscopy. The results obtained in this sensor chip show no difference from those obtained on a regular grating-coupled SPR sensor chip.     

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.     

Biocompatible surface preparation using amino-functionalized amylose

Aminopropyl amyloses with various degrees of substitution (DS) were prepared and investigated with respect to their surface modification properties. Poly(acrylic acid) was grafted to plasma-activated PVDF films, and the functional amylose was bound via amide linkage formation. Layer formation was confirmed by X-ray photoelectron spectroscopy. Contact angle measurements and surface MALDI-TOF mass spectrometry indicated a hydrophilic surface and minimization of protein adsorption.     

Impact of spacers on the hybridization efficiency of mixed self-assembled DNA/alkanethiol films

The immobilization of DNA strands is an essential step in the development of any DNA biosensor. Self-assembled mixed DNA/alkanethiol films are often used for coupling DNA probes covalently to the sensor surface. Although this strategy is well accepted, the effect of introducing a spacer molecule to increase the distance between the specific DNA sequence and the surface has rarely been assessed. The major goal of this work was to evaluate a number of such spacers and to assess their impact on for example the sensitivity and the reproducibility. Besides the commonly used mercaptohexyl (C(6)) spacer, a longer mercapto-undecyl (C(11)) spacer was selected. The combination of both spacers with tri(ethylene)glycol (TEG) and hexa(ethylene)glycol (HEG) was studied as well. The effect of the different spacers on the immobilization degree as well as on the consecutive hybridization was studied using surface plasmon resonance (SPR). When using the longer C(11) spacer the mixed DNA/alkanethiol films were found to be more densely packed. Further hybridization studies have indicated that C(11) modified probes improve the sensitivity, the corresponding detection limit as well as the reproducibility. In addition two different immobilization pathways, i.e. flow vs. diffusion controlled, were compared with respect to the hybridization efficiency. These data suggest that a flow-assisted approach is beneficial for DNA immobilization and hybridization events. In conclusion, this work demonstrates the considerable impact of spacers on the biosensor performance but also shows the importance of a flow-assisted immobilization approach.     

Adsorption of plasmid DNA onto N,N'- (dimethylamino)ethyl-methacrylate graft-polymerized poly-L-lactic acid film surface for promotion of in-situ gene delivery

The surface of biodegradable poly-L-lactic acid (PLLA) film was modified with N,N'-(dimethylamino)ethyl-methacrylate (DMAEMA) via UV-induced graft copolymerization, and plasmid DNA molecules were adsorbed onto the surface of modified PLLA film by electrostatic interactions with cationic DMAEMA polymer. We characterized the structure of the modified PLLA film surface by Fourier transform infrared attenuated total reflection (FTIR-ATR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The weight-average molecular weight (Mw) of grafted DMAEMA polymer chains was estimated from the elution time of gel filtration chromatography. C.I. Acid Orange 7 dyeing results indicated that graft density of DMAEMA on PLLA film increased with the UV irradiation time and then reached a saturated value. DNA adsorption density was proportioned to graft density of DMAEMA. Mouse fibroblast L929 cell line was cultured on modified PLLA films, and cell viability and gene transfection efficiency were monitored after 2 days culture. It was found that the DMAEMA grafted PLLA film had obvious cytotoxicity to the cells. On the contrary, cytotoxicity of the surface was highly decreased after adsorption with plasmid DNA. This DNA adsorbed DMAEMA modified PLLA showed the ability to deliver DNA into mammalian cells cultured on the surface with high-transfection efficiency at a low DNA amount. The present results suggest that the DMAEMA grafted PLLA has potentiality to be used as a safe and effective gene delivery system in gene-activated materials.     

Structure and organization of the microsomal xenobiotic epoxide hydrolase gene

The gene for the microsomal xenobiotic rat liver epoxide hydrolase has been isolated and characterized. Clones were obtained from a Wistar Furth Charon 35 genomic library by hybridization with a full-length epoxide hydrolase cDNA. The gene for the xenobiotic epoxide hydrolase is approximately 16 kilobases in length and consists of 9 exons ranging in size from 109 to 420 base pairs and 8 intervening sequences, the largest of which is 3.2 kilobases. S1-nuclease mapping, primer extension studies, and sequence analysis were used to determine the 5' cap site and the size of the first exon (170 base pairs). Regulatory sequences analogous to TATA, CCAAT, and core enhancer sequences were noted in the 5'-flanking region of the gene. The cDNA and gene for epoxide hydrolase displayed nucleotide sequence identity although they were isolated from different rat strains. Also, Southern blot analysis of restricted liver DNA from inbred Fischer 344 and Wistar Furth rat strains, and outbred Sprague-Dawley rats indicated a high degree of structural similarity for the epoxide hydrolase gene within these three strains. Only a single functional epoxide hydrolase gene was identified and no evidence of hybridization to the genes for the microsomal cholesterol epoxide hydrolase or the cytosolic epoxide hydrolase was observed. However, a pseudogene for the microsomal xenobiotic epoxide hydrolase was isolated and characterized from the genomic library.     

Microgravimetric DNA sensor based on quartz crystal microbalance: comparison of oligonucleotide immobilization methods and the application in genetic diagnosis

We report on the study of immobilization DNA probes onto quartz crystal oscillators by self-assembly technique to form variety types of mono- and multi-layered sensing films towards the realization of DNA diagnostic devices. A 18-mer DNA probe complementary to the site of genetic beta-thalassaemia mutations was immobilized on the electrodes of QCM by covalent bonding or electrostatic adsorption on polyelectrolyte films to form mono- or multi-layered sensing films by self-assembled process. Hybridization was induced by exposure of the QCMs immobilized with DNA probe to a test solution containing the target nucleic acid sequences. The kinetics of DNA probe immobilization and hybridization with the fabricated DNA sensors were studied via in-situ frequency changes. The characteristics of QCM sensors containing mono- or multi-layered DNA probe constructed by direct chemical bonding, avidin-biotin interaction or electrostatic adsorption on polyelectrolyte films were compared. Results indicated that the DNA sensing films fabricated by immobilization of biotinylated DNA probe to avidin provide fast sensor response and high hybridization efficiencies. The effects of ionic strength of the buffer solution and the concentration of target nucleic acid used in hybridization were also studied. The fabricated DNA biosensor was used to detect a set of real samples. We conclude that the microgravimetric DNA sensor with its direct detection of amplified products provide a rapid, low cost and convenient diagnostic method for genetic disease.     

DNA immobilization/hybridization on plasma-polymerized pyrrole

The present work describes the fabrication and characterization of the conducting polymer coatings prepared by the continuous wave plasma polymerization and the applications as adhesion layers for studying DNA immobilization/hybridization. The stability of plasma polymerized pyrrole (ppPY) in the aqueous solution was characterized by ellipsometry. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy were used to investigate polymer matrix properties and oligonucleotide/DNA binding interaction. The successful DNA immobilization on ppPY surfaces was found to depend on the macromolecular architecture of plasma polymerized films. The plasma polymers with similar thickness deposited at different input powers showed various comparable immobilization properties. The plasma-polymerized films prepared at the low input power showed a lower sensitivity toward DNA binding than those films deposited at the high input power. This result will be important to study plasma polymerized films as potential DNA biosensors in the future.     

Synthesis and genotoxicity of acetoxyoxirane, the epoxide of vinyl acetate

Acetoxyoxirane, the epoxide of vinyl acetate and a potential reactive intermediate, was synthesized and characterized by 13C-nuclear magnetic resonance (13C-NMR) and mass spectroscopy. The compound induced lesions (endonuclease-sensitive and alkali-labile sites) in supercoiled PM2 DNA in vitro and was directly mutagenic toward Salmonella typhimurium TA100. The mutagenicity of the epoxide in phosphate buffer (pH 7.4, 37 degrees C) decreased, with an initial half-life of 2.8 minutes, and mutagenicity was completely abolished by addition of S-9 mix. Acetoxyoxirane did not induce unscheduled DNA synthesis on incubation with Syrian hamster embryo fibroblasts (SHE cells). These findings may possibly be explained by an effective inactivation of acetoxyoxirane by esterases when these are present in the biological system. This view is consistent with the lack of acetoxyoxirane detected in rat liver microsomal incubations of vinyl acetate.     

Covalent DNA immobilization on polymer-shielded silver-coated quartz crystal microbalance using photobiotin-based UV irradiation.

The use of a commercial, silver-coated quartz crystal microbalance (QCM) as a disposable, low-cost, and reliable DNA sensor is presented. This is an incorporation of polymer-based silver electrode shielding and photochemistry-based surface modification for covalent DNA immobilization. To prevent undesired oxidation, the silver electrodes are coated with thin polystyrene films. The polymer surfaces are then modified by a photoreactive biotin derivative (photobiotin) under UV irradiation. The resulting biotin residues on the polymer-shielded surface react with a tetrameric avidin. Consequently a biotin-labeled DNA probe can be immobilized through a biotin-avidin-biotin bridge. A 14-mer single-stranded biotin-DNA probe and a 70-mer single-stranded DNA fragment containing complementary or noncomplementary sequences are used as a model system for DNA hybridization assay on the proposed sensors. The shielding ability of the polystyrene coatings after photo irradiation is investigated. The DNA probe binding capacity, hybridization efficiency, and kinetics are also investigated.     

Label-free electrochemical DNA sensor based on functionalised conducting copolymer

A simple and label-free electrochemical sensor for recognition of the DNA hybridization event was prepared based on a new functionalised conducting copolymer, poly[pyrrole-co-4-(3-pyrrolyl) butanoic acid]. This precursor copolymer can be easily electrodeposited on the electrode surface and shows high electroactivity in an aqueous medium. An amino-substituted oligonucleotide (ODN) probe was covalently grafted onto the surface of the copolymer in a one step procedure and tested on hybridization with complementary ODN segments. The cyclic voltammogram of ODN probe-modified copolymer showed very little change when incubated in presence of non-complementary ODN, while a significant, and reproducible, modification of the voltammogram was observed after addition of complementary ODN. The AC impedance spectrum showed an increased charge transfer resistance (Rct) and double layer capacitance of the sensor film after hybridisation. Sensors with thinner films showed higher sensitivity than thicker films, suggesting that hybridisation at or near the surface of the film produces a larger change in electrical properties than that within the body of the film.