Plasma Polymer Film Structure and DNA Probe Immobilization

Analysis of the molecular interaction of glycosylated proteins with rabbit liver asialoglycoprotein receptors using surface plasmon resonance spectroscopy

This research aims to develop DNA chip array without an indicator. We fabricated microelectrode array by photolithography technology. Several DNA probes were immobilized on an electrode. Then, indicator-free target DNA was hybridized by an electrical force and measured electrochemically. Cyclic-voltammograms (CVs) showed a difference between DNA probe and mismatched DNA in an anodic peak. Immobilization of probe DNA and hybridization of target DNA could be confirmed by fluorescent. This indicator-free DNA chip microarray resulted in the sequence-specific detection of the target DNA quantitatively ranging from <TEX>$10^{-18}\;M\;to\;10^{-5}$</TEX> M in the buffer solution. This indicator-free DNA chip resulted in a sequence-specific detection of the target DNA.

Detection of disease-related gene expression by DNA hybridization is a useful diagnostic method. In this study a monolayer graphene field effect transistor (GFET) was fabricated for the detection of a particular single-stranded DNA (target DNA). The probe DNA, which is a single-stranded DNA with a complementary nucleotide sequence, was directly immobilized onto the graphene surface without any linker. The VDirac was shifted to the negative direction in the probe DNA immobilization. A further shift of VDirac in the negative direction was observed when the target DNA was applied to GFET, but no shift was observed upon the application of non-complementary mismatched DNA. Direct immobilization of double-stranded DNA onto the graphene surface also shifted the VDirac in the negative direction to the same extent as that of the shift induced by the immobilization of probe DNA and following target DNA application. These results suggest that the further shift of VDirac after application of the target DNA to the GFET was caused by the hybridization between the probe DNA and target DNA.

Development of DNA electrochemical biosensor based on immobilization of ssDNA on the surface of nickel oxide nanoparticles modified glassy carbon electrode

A direct electrochemical detection procedure for DNA hybridization by using the electrochemical signal changes of conductive poly(m-aminobenzenesulfonic) acid (PABSA)/TiO(2) nanosheet membranes, which were electropolymerized by using the pulse potentiostatic method, is reported. Due to the unique properties of TiO(2) nanoparticles, m-aminobenzenesulfonic acid monomers tend to be adsorbed around the particles, and the electropolymerization efficiency is greatly improved. The combination of TiO(2) nanoparticles and PABSA resulted in a nanocomposite membrane with unique and novel nanosheet morphology that provides more activation sites and enhances the surface electron-transfer rate. These characteristics were propitious for the magnification of PABSA electrochemical signals and the direct detection of DNA hybridization. Owing to the presence of abundant sulfonic acid groups, PABSA could overcome the drawbacks of polyaniline and be used to detect bioanalytes at physiological pH. DNA probes could be covalently attached to the sulfonic groups through the amines of DNA sequences by using an acyl chloride cross-linking reaction. After immobilization of probe DNA, the electrochemical impedance value increased significantly compared to that of PABSA/TiO(2) nanosheet membranes, and then decreased dramatically after the hybridization reaction of the probe DNA with the complementary DNA sequence compared to that of the probe-immobilized electrode. Electrochemical impedance spectroscopy was adopted for indicator-free DNA biosensing, which had an eminent ability for the recognition between double-base mismatched sequences or non-complementary DNA sequences and complementary DNA sequences. A gene fragment, which is related to one of the screening genes for the transgenically modified plants, the cauliflower mosaic virus 35S gene was satisfactorily detected. This is the first report for the indicator-free impedance DNA hybridization detection by using PABSA/TiO(2) membranes under neutral conditions.

Thin films of plasma polymerized pentafluorophenyl methacrylate (pp-PFM) offer highly reactive ester groups throughout the structure of the film that allow for subsequent reactions with different aminated reagents and biological molecules. The present paper follows on from previous work on the plasma deposition of pentafluorophenyl methacrylate (PFM) for optimum functional group retention (Francesch, L.; Borros, S.; Knoll, W.; Foerch, R. Langmuir 2007, 23, 3927) and reactivity in aqueous solution (Duque, L.; Queralto, N.; Francesch, L.; Bumbu, G. G.; Borros, S.; Berger, R.; Förch, R. Plasma Process. Polym. 2010, accepted for publication) to investigate the binding of a biologically active peptide known to induce cellular adhesion (IKVAV) and of biochemically active proteins such as BSA and fibrinogen. Analyses of the films and of the immobilization of the biomolecules were carried out using infrared reflection absorption spectroscopy (IRRAS), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). The attachment of the biomolecules on pulsed plasma polymerized pentafluorophenyl methacrylate was monitored using surface plasmon resonance spectroscopy (SPR). SPR analysis confirmed the presence of immobilized biomolecules on the plasma polymer and was used to determine the mass coverage of the peptide and proteins adsorbed onto the films. The combined analysis of the surfaces suggests the covalent binding of the peptide and proteins to the surface of the pp-PFM.

A proof of concept procedure for the electroaddressable covalent immobilization of DNA and protein on arrayed electrodes along with simultaneous detection of multiple bioagents in the same sample solution is described. Carboxyphenyldiazonium was selectively deposited onto five of nine individually addressable electrodes in an array via bias assisted assembly. Amine functionalized DNA probes were covalently coupled to the carboxyl surface via carbodiimide chemistry. This was followed by the covalent immobilization of diazonium-antibody conjugates into the remaining four electrodes via cyclic voltammetry. Simultaneous electrochemical detection of a DNA sequence related to the breast cancer BRCA1 gene and the human cytokine protein interleukin-12, which is a substantial component in the immune system response and attack of tumor cells, is reported. These results demonstrate the possibility of selective patterning of diverse biomolecules on a single device and may have significant implications for future development of microarrays and biosensors.

Plasma deposited polymer films from simple organic molecules with a polymerizeable C=C bond, styrene, ethylene, allylamine, and allyl alcohol, were studied by Time of Flight Static Secondary Ion Mass Spectrometry (ToF- SSIMS) before and after exposure to ambient air. ToF-SSIMS analysis of the plasma deposited polymer films was performed immediately after their preparation without any air contact, i.e., under so called in-situ conditions. The influence of the external plasma parameters, duty cycle, plasma power, monomer flow rate, and reaction pressure, on the secondary ion mass spectra of plasma deposited films was investigated. From these data, information on the chemical character of the surface of plasma deposited films was derived. In detail, the unsaturated, branched and cross-linked character of the plasma polymers was described. The problem of the retention of relevant structure units, e.g., functional groups, in plasma deposited polymers, was elucidated in detail using SSIMS data. Harder plasma conditions, which can be obtained when high duty cycles, high plasma power, and low monomer flow rates are employed, result in an increase in the unsaturated, branched and/or cross- linked character of the investigated plasma polymers. Additionally, the retention of the relevant structure groups, e.g., aromatic rings in the case of styrene and side chain groups in the case of allylamine and allyl alcohol plasma polymers, decreases when the plasma conditions become harder. Plasma polymers with high functional group retention and a minimum degree of unsaturation and branching and cross-linking were obtained at the mildest plasma conditions applied in this study. Plasma copolymerization was investigated by using allyl alcohol, or allylamine as functional group bearing monomers and styrene, or ethylene as chain extending monomers. It was found that through a careful selection of the feed gas composition the concentration of surface functional groups in plasma deposited films can be controlled. In addition, the ToF-SSIMS spectra of the plasma deposited copolymer films were found to be partially different from a superposition of the spectra of the respective plasma homopolymers. This suggests that interactions and recombination reactions are taking place between the monomer molecules in the plasma or during their respective plasma deposition process. Since the plasma polymers contain radical sites, extensive oxygen incorporation occurs when they are exposed to ambient air. It was found that the radical density of the plasma polymers and their ageing behavior is influenced by the nature of the monomer and the external plasma parameters. In the case of plasma deposited polymers prepared from olefinic and aromatic monomers, ethylene and styrene, respectively, a relation between the regularity of the plasma polymers and the amount of oxygen incorporation was found. The oxygen uptake of these plasma polymers increases when the plasma conditions become harder. On the contrary, the oxygen uptake of the plasma deposited allylamine films did not depend on the external plasma parameters. In the case of plasma deposited allyl alcohol films, it was found that their ageing on air is probably defined by two competing processes, i.e. the oxygen uptake and the diffusion of low molecular weight oxidized species into the bulk together with re-orientations of oxygen functional groups. The ageing behavior of plasma deposited copolymers films was found to be dependent on the feed gas composition.

Plasma polymerization on a metal substrate using DC glow discharge was carried out for forming an adherent layer using a hydrocarbon reactive monomer as a function of deposition time, voltage, current, and pressure. Strong adhesion of the polymerized film to the metal and high surface energy were achieved at optimum plasma discharge conditions. The surface and the interface of the polymer film were investigated by Fourier transformation Infrared (FT-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The FT-IR spectra of the polymers obtained by DC glow discharge are similar to those of the polymers obtained by RF discharge, but good adhesion of the polymer films to the metal could be obtained only by the DC discharge polymerization. The factors affecting the adhesion such as polymer damage, functional groups, and discharge conditions are investigated. Applications of the adherent layer prepared by plasma polymerization are also presented.

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.

Microfluidic system for DNA sequence detection

Programmable graphene-based microfluidic sensor for DNA detection

Effect of aqueous solution on functional plasma polymerized films

This contribution reports on a novel self-assembled monolayer (SAM) on a gold surface, able to immobilize double-strand DNA and to induce its cleavage upon light excitation. DNA binding with the SAM is mainly driven by a highly favored electrostatic interaction, and it is shown that DNA immobilization takes place without impairing its intrinsic high order. SAM irradiation leads to an uncommon defluorination reaction, and it is unambiguously demonstrated that DNA breakage is strictly interrelated to this photochemical pathway. The photocleavage process is very efficient, is exclusively controlled by illumination conditions, seems to be non-base-specific, and does not require external additives. These features, together with the low excitation energy required, the prolonged thermal stability under physiological conditions, and the ease of preparation, make the SAM presented herein an appealing model system for designing nanodevices for applications where efficient, non-base-specific, and externally tunable cleavage of nanoscaled DNA arrays is required.

Development of an ultrasensitive electrochemical genosensor for detection of HIV-1 pol gene using a gold nanoparticles coated carbon paste electrode impregnated with lead ion-imprinted polymer nanomaterials as a novel electrochemical probe