Molecularly imprinted Au nanoparticles composites on Au surfaces for the surface plasmon resonance detection of pentaerythritol tetranitrate, nitroglycerin, and ethylene glycol dinitrate.

Molecularly imprinted Au nanoparticles (NPs) composites are generated on Au-coated glass surfaces. The imprinting process involves the electropolymerization of thioaniline-functionalized Au NPs (3.5 nm) on a thioaniline monolayer-modified Au surface in the presence of a carboxylic acid, acting as a template analogue for the respective explosive. The exclusion of the imprinting template from the Au NPs matrix yields the respective imprinted composites. The binding of the analyte explosives to the Au NPs matrixes is probed by surface plasmon resonance spectroscopy, SPR, where the electronic coupling between the localized plasmon of the Au NPs and the surface plasmon wave leads to the amplification of the SPR responses originating from the dielectric changes of the matrixes upon binding of the different explosive materials. The resulting imprinted matrixes reveal high affinities and selectivity toward the imprinted explosives. Using citric acid as an imprinting template, Au NPs matrixes for the specific analysis of pentaerythritol tetranitrate (PETN) or of nitroglycerin (NG) were prepared, leading to detection limits of 200 fM and 20 pM, respectively. Similarly, using maleic acid or fumaric acid as imprinting templates, high-affinity sensing composites for ethylene glycol dinitrate (EGDN) were synthesized, leading to a detection limit of 400 fM for both matrixes.

Deposition of functionalized polymer layers in surface plasmon resonance immunosensors by in-situ polymerization in the evanescent wave field.

Traditionally, the integration of sensing gel layers in surface plasmon resonance (SPR) is achieved via "bulk" methods, such as precipitation, spin-coating or in-situ polymerization onto the total surface of the sensor chip, combined with covalent attachment of the antibody or receptor to the gel surface. This is wasteful in terms of materials as the sensing only occurs at the point of resonance interrogated by the laser. By isolating the sensing materials (antibodies, enzymes, aptamers, polymers, MIPs, etc.) to this exact spot a more efficient use of these recognition elements will be achieved. Here we present a method for the in-situ formation of polymers, using the energy of the evanescent wave field on the surface of an SPR device, specifically localized at the point of interrogation. Using the photo-initiator couple of methylene blue (sensitizing dye) and sodium p-toluenesulfinate (reducing agent) we polymerized a mixture of N,N-methylene-bis-acrylamide and methacrylic acid in water at the focal point of SPR. No polymerization was seen in solution or at any other sites on the sensor surface. Varying parameters such as monomer concentration and exposure time allowed precise control over the polymer thickness (from 20-200 nm). Standard coupling with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide was used for the immobilization of protein G which was used to bind IgG in a typical biosensor format. This model system demonstrated the characteristic performance for this type of immunosensor, validating our deposition method.

Stimulation of human olfactory receptor 17-40 with odorants probed by surface plasmon resonance.

We report here the results of human olfactory receptor (OR) 17-40 stimulation with some odorants probed by means of the double-channel surface plasmon resonance platform NanoSPR-6. OR 17-40 tagged with N-terminal cmyc sequence was heterologously co-expressed with Galpha(olf) protein in yeast, and receptor-carrying nanosomes were prepared from yeast membrane fraction. Then, receptors were specifically captured via anti-cmyc antibody attached to the gold-coated substrate in orientated or random way. Measurement of odorants effects were carried out in the presence of GTP-gamma-S in differential mode in order to compensate bulk changes of refractive index. For the first time, biosensing efficiency of olfactory films was discussed in terms of their thickness and Galpha(olf) accessibility to GTP-gamma-S. Bell-shaped response profile with two maxima (near 1 nM and near 1 microM) was established for helional, which is documented as highly specific agonist of OR 17-40. Unrelated odorant heptanal used as control, did not evoke significant variations of differential signal.

Switchable surface properties through the electrochemical or biocatalytic generation of Ag0 nanoclusters on monolayer-functionalized electrodes.

The electroswitchable and the biocatalytic/electrochemical switchable interfacial properties of a Ag(+)-biphenyldithiol (BPDT) monolayer associated with a Au surface are described. Upon the application of a potential corresponding to -0.2 V the Ag(+)-BPDT is reduced to the Ag(0)-BPDT interface, and silver nanoclusters are generated on the interface. The application of a potential that corresponds to 0.2 V reoxidizes the monolayer to the Ag(+)-BPDT monolayer. The reversible electrochemical transformation of the Ag(+)-BPDT monolayer and of the Ag(0)-BPDT surface was followed by electrochemical means and surface plasmon resonance spectroscopy (SPR). The SPR experiments enabled us to follow the kinetics of nanoclustering of Ag(0) on the surface. The hydrophobic/hydrophilic properties of the surface are controlled by the electrochemically induced transformation of the interface between the Ag(+)-BPDT and Ag(0)-BPDT states. The Ag(0)-BPDT monolayer reveals enhanced hydrophilicity. The hydrophobic/hydrophilic properties of the interface were probed by contact angle measurements and force interactions with a hydrophobically-functionalized AFM tip. The Ag(0)-BPDT interface was also biocatalytically generated using alkaline phosphatase, AlkPh, and p-aminophenyl phosphate as substrate. The biocatalytically generated p-aminophenol reduces Ag(+) ions associated with the surface to Ag(0) nanoclusters. This enables the cyclic biocatalytic/electrochemical control of the surface properties of the modified electrode.

Enzyme-catalyzed bio-pumping of electrons into au-nanoparticles: a surface plasmon resonance and electrochemical study.

The enzyme glucose oxidase (GOx) is reconstituted on a flavin adenin dinucleotide (FAD, 1) cofactor-functionalized Au-nanoparticle (Au-NP), 1.4 nm, and the GOx/Au-NP hybrid is linked to a bulk Au-electrode by a short dithiol, 1,4-benzenedithiol (2), or a long dithiol, 1,9-nonanedithiol (3), monolayer. The reconstituted GOx/Au-NP hybrid system exhibits electrical communication between the enzyme redox cofactor and the Au-NP core. Because the thiol monolayers provide a barrier for electron tunneling, the electron transfer occurring upon the biocatalytic oxidation of glucose results in the Au-NPs charging. The charging of the Au-NPs alters the plasma frequency and the dielectric constant of the Au-NPs, thus leading to the changes of the dielectric constant of the interface. These are reflected in pronounced shifts of the plasmon angle, theta(P), in the surface plasmon resonance (SPR) spectra. As the biocatalytic charging phenomenon is controlled by the concentration of glucose, the changes in the theta(P) values correlate with the concentration of glucose. The biocatalytic charging process is characterized by following the differential capacitance of the GOx/Au-NP interface and by monitoring the potential generated on the bulk Au-electrode. The charging of the GOx/Au-NPs is also accomplished in the absence of glucose by the application of an external potential on the electrode, that resulted in similar plasmon angle shifts. The results allowed us to estimate the number of electrons stored per Au-NP at variable concentrations of glucose in the presence of the two different thiol linkers.

Probing antigen-antibody binding processes by impedance measurements on ion-sensitive field-effect transistor devices and complementary surface plasmon resonance analyses: development of cholera toxin sensors.

Impedance measurements on ISFET devices are employed to develop new immunosensors. The analysis of the transconductance curves recorded at variable frequencies, upon the formation of antigen-antibody complexes on the ISFET devices, allows determination of the biomaterial film thicknesses. Complementary surface plasmon resonance measurements of analogous biosensor systems, using Au-coated glass slides as support, reveal similar film thicknesses of the biomaterials and comparable detection limits. A dinitrophenyl antigen layer is immobilized on the ISFET gate as a sensing interface for the anti-dinitrophenyl antibody (anti-DNP-Ab). The anti-DNP-Ab is analyzed with a sensitivity that corresponds to 0.1 microg mL(-1). The assembly of the biotinylated anti-anti-DNP-Ab and avidin layers on the base anti-DNP-Ab layer is characterized by impedance measurements. The development of an ISFET-based sensor for the cholera toxin is described. The anti-cholera toxin antibody is immobilized on the ISFET device. The association of the cholera toxin (CT) to the antibody is monitored by the impedance measurements. The detection limit for analyzing CT is 1.0 x 10(-11) M.