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1.  2D-SEIRA spectroscopy to highlight conformational changes of the cytochrome c oxidase induced by direct electron transfer†‡ 
Potentiometric titrations of the cytochrome c oxidase (CcO) immobilized in a biomimetic membrane system were followed by two-dimensional surface-enhanced IR absorption spectroscopy (2D SEIRAS) in the ATR-mode. Direct electron transfer was employed to vary the redox state of the enzyme. The CcO was shown to undergo a conformational transition from a non-activated to an activated state after it was allowed to turnover in the presence of oxygen. Differences between the non-activated and activated state were revealed by 2D SEIRA spectra recorded as a function of potential. The activated state was characterized by a higher number of correlated transitions as well as a higher number of amino acids associated with electron transfer.
doi:10.1039/c0mt00083c
PMCID: PMC3912184  PMID: 21541411
2.  Two-Dimensional Heterospectral Correlation Analysis of the Redox-Induced Conformational Transition in Cytochrome c Using Surface-Enhanced Raman and Infrared Absorption Spectroscopies on a Two-Layer Gold Surface 
The Journal of Physical Chemistry. B  2013;117(33):9606-9614.
The heme protein cytochrome c adsorbed to a two-layer gold surface modified with a self-assembled monolayer of 2-mercaptoethanol was analyzed using a two-dimensional (2D) heterospectral correlation analysis that combined surface-enhanced infrared absorption spectroscopy (SEIRAS) and surface-enhanced Raman spectroscopy (SERS). Stepwise increasing electric potentials were applied to alter the redox state of the protein and to induce conformational changes within the protein backbone. We demonstrate herein that 2D heterospectral correlation analysis is a particularly suitable and useful technique for the study of heme-containing proteins as the two spectroscopies address different portions of the protein. Thus, by correlating SERS and SEIRAS data in a 2D plot, we can obtain a deeper understanding of the conformational changes occurring at the redox center and in the supporting protein backbone during the electron transfer process. The correlation analyses are complemented by molecular dynamics calculations to explore the intramolecular interactions.
doi:10.1021/jp404573q
PMCID: PMC3753128  PMID: 23930980
3.  Active Control of SPR by Thermoresponsive Hydrogels for Biosensor Applications 
The use of thermoresponsive poly(N-isopropylacrylamide)-based hydrogel (pNIPAAm) for rapid tuning of surface plasmon resonance (SPR) is reported. This approach is implemented by using an SPR layer architecture with an embedded indium tin oxide microheater and pNIPAAm film on its top. It takes advantage of rapid thermally induced swelling and collapse of pNIPAAm that is accompanied by large refractive index changes and leads to high thermo-optical coefficient of dn/dT = 2 × 10–2 RIU/K. We show that this material is excellently suited for efficient control of refractive index-sensitive SPR and that it can serve simultaneously as a 3D binding matrix in biosensor applications (if modified with biomolecular recognition elements for a specific capture of target analyte). We demonstrate that this approach enables modulating of the output signal in surface plasmon-enhanced fluorescence spectroscopy biosensors and holds potential for simple time-multiplexing of sensing channels for parallelized readout of fluorescence assays.
doi:10.1021/jp400255u
PMCID: PMC3677233  PMID: 23762499
4.  Macromolecular shape and interactions in layer-by-layer assemblies within cylindrical nanopores 
Summary
Layer-by-layer (LbL) deposition of polyelectrolytes and proteins within the cylindrical nanopores of anodic aluminum oxide (AAO) membranes was studied by optical waveguide spectroscopy (OWS). AAO has aligned cylindrical, nonintersecting pores with a defined pore diameter d 0 and functions as a planar optical waveguide so as to monitor, in situ, the LbL process by OWS. The LbL deposition of globular proteins, i.e., avidin and biotinylated bovine serum albumin was compared with that of linear polyelectrolytes (linear-PEs), both species being of similar molecular weight. LbL deposition within the cylindrical AAO geometry for different pore diameters (d 0 = 25–80 nm) for the various macromolecular species, showed that the multilayer film growth was inhibited at different maximum numbers of LbL steps (n max). The value of n max was greatest for linear-PEs, while proteins had a lower value. The cylindrical pore geometry imposes a physical limit to LbL growth such that n max is strongly dependent on the overall internal structure of the LbL film. For all macromolecular species, deposition was inhibited in native AAO, having pores of d 0 = 25–30 nm. Both, OWS and scanning electron microscopy showed that LbL growth in larger AAO pores (d 0 > 25–30 nm) became inhibited when approaching a pore diameter of d eff,n_max = 25–35 nm, a similar size to that of native AAO pores, with d 0 = 25–30 nm. For a reasonable estimation of d eff,n_max, the actual volume occupied by a macromolecular assembly must be taken into consideration. The results clearly show that electrostatic LbL allowed for compact macromolecular layers, whereas proteins formed loosely packed multilayers.
doi:10.3762/bjnano.3.54
PMCID: PMC3458591  PMID: 23019541
avidin-biotin; dendrimers; nanoporous substrates; optical lightmode waveguide spectroscopy; polyelectrolytes
5.  Electrochemical Surface Plasmon Resonance (EC-SPR) and Waveguide Enhanced Glucose Biosensing with N-Alkylaminated Polypyrrole/Glucose Oxidase Multilayers 
ACS applied materials & interfaces  2010;2(8):2347-2354.
In this work, we report an electrochemical surface plasmon resonance/waveguide (EC-SPR/waveguide) glucose biosensor, which could detect enzymatic reactions in a conducting polymer/glucose oxidase (GOx) multilayer thin film. In order to achieve a controlled enzyme electrode and waveguide mode, GOx (negatively charged) was immobilized with a water-soluble conducting N-alkylaminated polypyrrole (positively charged) using the layer-by-layer (LbL) electrostatic self-assembly technique. The electrochemical and optical signals were simultaneously obtained from the composite LbL enzyme electrode upon addition of glucose as mediated by the electroactivity and electrochromic property of the polypyrrole layers. The signal enhancement in the EC-SPR detection is obtained by monitoring the doping-dedoping events on the polypyrrole. The real time optical signal could be distinguished between the change in the dielectric constant of the enzyme layer and other non-enzymatic reaction events such as adsorption of glucose and change of refractive index of solution. This was possible by a correlation of both the SPR mode, m=0, and m=1 mode of the waveguide in an SPR/waveguide spectroscopy experiment.
doi:10.1021/am100373v
PMCID: PMC2929602  PMID: 20666478
surface plasmon; glucose; biosensor; conducting polymer; enzyme; waveguide
6.  Surface plasmon optical study of the interfacial phase transition of elastinlike polypeptide grafted on gold 
Biointerphases  2008;3(3):66-74.
The conformational changes in elastinlike polypeptides (ELPs) grafted to a solid/solution interface via different architectures were studied using surface plasmon resonance spectroscopy and surface plasmon field-enhanced fluorescence spectroscopy (SPFS). SPFS provides a simple and convenient optical method to study the influence of the grafting method and the graft density on the conformational changes in ELPs at the solid-solution interface as a function of environmental variables. A typical response of the ELP, consistent with its stimuli responsiveness, was a gradual collapse upon increasing the ionic strength; this effect was inversely correlated with the surface graft density of the ELP.
doi:10.1116/1.2965133
PMCID: PMC2856947  PMID: 20408702
7.  Triangular neuronal networks on microelectrode arrays: an approach to improve the properties of low-density networks for extracellular recording 
Biomedical Microdevices  2009;11(6):1269-1278.
Multi-unit recording from neuronal networks cultured on microelectrode arrays (MEAs) is a widely used approach to achieve basic understanding of network properties, as well as the realization of cell-based biosensors. However, network formation is random under primary culture conditions, and the cellular arrangement often performs an insufficient fit to the electrode positions. This results in the successful recording of only a small fraction of cells. One possible approach to overcome this limitation is to raise the number of cells on the MEA, thereby accepting an increased complexity of the network. In this study, we followed an alternative strategy to increase the portion of neurons located at the electrodes by designing a network in confined geometries. Guided settlement and outgrowth of neurons is accomplished by taking control over the adhesive properties of the MEA surface. Using microcontact printing a triangular two-dimensional pattern of the adhesion promoter poly-D-lysine was applied to the MEA offering a meshwork that at the same time provides adhesion points for cell bodies matching the electrode positions and gives frequent branching points for dendrites and axons. Low density neocortical networks cultivated under this condition displayed similar properties to random networks with respect to the cellular morphology but had a threefold higher electrode coverage. Electrical activity was dominated by periodic burst firing that could pharmacologically be modulated. Geometry of the network and electrical properties of the patterned cultures were reproducible and displayed long-term stability making the combination of surface structuring and multi-site recording a promising tool for biosensor applications.
doi:10.1007/s10544-009-9346-0
PMCID: PMC2776171  PMID: 19757074
Cell patterning; Neuronal networks; Microelectrode arrays; Microcontact printing; Cell-based Biosensors; Burst firing
8.  A Comparative Plasmonic Study of Nanoporous and Evaporated Gold Films 
Plasmonics (Norwell, Mass.)  2008;3(1):13-20.
Previously, we have reported that nanoporous gold (NPG) films prepared by a chemical dealloying method have distinctive plasmonic properties, i.e., they can simultaneously support localized and propagating surface plasmon resonance modes (l-SPR and p-SPR, respectively). In this study, the plasmonic properties of NPG are quantified through direct comparison with thermally evaporated gold (EG) films. Cyclic voltammetry and electrochemical impedance spectroscopy experiments reveal that the NPG films have 4–8.5 times more accessible surface area than EG films. Assemblies of streptavidin–latex beads generate p-SPR responses on both NPG and EG films that correlate well with the bead density obtained from scanning electron microscopy (SEM) images. A layer-by-layer assembly experiment on NPG involving biotinylated anti-avidin IgG and avidin, studied by l-SPR and SEM, shows that the l-SPR signal is directly linked to the accessibility of the interior of the NPG porosity, an adjustable experimental parameter that can be set by the dealloying condition and time.
Electronic supplementary material
The online version of this article (doi:10.1007/s11468-007-9048-5) contains supplementary material, which is available to authorized users.
doi:10.1007/s11468-007-9048-5
PMCID: PMC2758361  PMID: 19816537
Surface plasmon resonance; Alloys; Layer deposition; Spectroscopy; Nanoporous gold; Gold films; Electrochemistry
9.  Surface plasmon field-enhanced fluorescence spectroscopy studies of primer extension reactions 
Nucleic Acids Research  2005;33(7):e69.
Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) utilizes the evanescent electromagnetic field of a surface plasmon to excite chromophors in close proximity to the surface. While conventional surface plasmon resonance spectroscopy allows the observation of surface reactions by means of refractive index changes, SPFS additionally provides a channel for the read-out of fluorescence changes. Thus, the detection limit for low mass compounds, whose adsorption is only accompanied by small refractive index changes, can be substantially improved by fluorescent labeling. In this study, we present the first example that utilizes SPFS to follow the dynamics of an enzymatic reaction. The elongation of surface-tethered DNA has been observed by the incorporation of Cy5-labeled nucleotides into the nascent strand by the action of DNA polymerase I (Klenow fragment). The technique offers a rapid way to determine the binding constant and the catalytic activity of a DNA processing enzyme, here exemplified by the Klenow fragment. Furthermore, the effect of mispaired bases in the primer/template duplex and the influence of different label densities have been studied. The resulting sensitivity for nucleotide incorporation, being in the femtomolar regime, combined with the specificity of the enzyme for fully complementary DNA duplexes suggest the application of this assay as a powerful tool for DNA detection.
doi:10.1093/nar/gni067
PMCID: PMC1084329  PMID: 15849312
10.  Surface plasmon field-enhanced fluorescence spectroscopy in PCR product analysis by peptide nucleic acid probes 
Nucleic Acids Research  2004;32(22):e177.
Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) was recently developed for PCR product analysis, which allowed for real-time monitoring of hybridization processes and for the detection of trace amounts of PCR products, with a detection limit of 100 fmol on the peptide nucleic acid (PNA) probe surface, and 500 fmol on the DNA probe surface. By selectively labeling the strands of PCR-amplified DNA, it was shown that the heat denaturation process in combination with the application of low-salt condition substantially reduced the interference from the antisense strands and thus simplified the surface hybridization. Furthermore, SPFS was demonstrated to be capable of quantitatively discriminating the difference induced by single nucleotide substitution, even within one minute of contact time.
doi:10.1093/nar/gnh175
PMCID: PMC545473  PMID: 15598819
11.  Oligonucleotide hybridization studied by a surface plasmon diffraction sensor (SPDS) 
Nucleic Acids Research  2004;32(9):e75.
A novel label-free biosensor concept based on surface plasmon-enhanced diffraction by micro- patterned interfaces was applied to the study of hybridization reactions of target DNA oligonucleotides (15mers and 75mers) from solution to probe DNA oligonucleotides attached via streptavidin to the sensor surface. The self-referencing and quadratic signal amplification mechanism of the sensor allowed highly sensitive detection of the hybridization process. Association and dissociation processes of DNA targets could be recorded in real time and used for the quantification of their binding affinities, which differ considerably with a single base pair mismatch. An equilibrium titration approach was also applied in order to obtain the binding affinities for 15mer targets, yielding similar affinity values. The hybridization efficiencies were found to be higher for the 15mers than for the 75mers, although the latter contained the same recognition sequences. The hybridization efficiency was shown to depend on the probe density and reached nearly 100% for the 15mer fully complementary targets at a probe density of ∼1.2 × 1012 molecules/cm2. Using the assay as an end-point determination method, the lowest detectable coverage of a 15mer oligonucleotide was at least ∼1.1 × 1011 molecules/cm2. The diffraction sensing concept offers a completely novel way to integrate a reference channel in large-scale, label-free screening applications, to improve the stability and to enhance the sensitivity of microarray read-out systems.
doi:10.1093/nar/gnh067
PMCID: PMC419622  PMID: 15155822
12.  Mismatching base-pair dependence of the kinetics of DNA–DNA hybridization studied by surface plasmon fluorescence spectroscopy 
Nucleic Acids Research  2004;32(8):2372-2377.
Two single-stranded DNAs consisting of complementary base pairs except for one mismatching base pair (MM1) can form double-stranded DNA by molecular recognition. This type of duplex is not as stable as that formed by MM0. In order to add to a better understanding of the physical mechanism of the hybridization and dissociation processes at sensor (chip) surfaces, we studied the kinetics of the MM1 hybridization by surface plasmon fluorescence spectroscopy. Target DNA strands labelled with a fluorescent molecule Cy5 at the 5′ end and hybridizing with the surface-attached probe DNA can be excited by the strong optical field of a surface plasmon resonance mode. The emitted fluorescence can be detected with high sensitivity. The affinity of a duplex was found to depend on the chemical nature, i.e. G–G, G–T etc., and on the position of the mismatching base pair along the 15mer duplex.
doi:10.1093/nar/gkh572
PMCID: PMC419460  PMID: 15115799

Results 1-12 (12)