Time-resolved surface enhanced resonance Raman spectro-electrochemistry of heme proteins

The membrane protein Cytochrome c Oxidase (CcO) is one of the most important functional bio-molecules. It appears in almost every eukaryotic cell and many bacteria. Although the different species differ in the number of subunits, the functional differences are merely marginal. CcO is the terminal link in the electron transfer pathway of the mitochondrial respiratory chain. Electrons transferred to the catalytic center of the enzyme conduce to the reduction of molecular oxygen to water. Oxygen reduction is coupled to the pumping of protons into the inter-membrane space and hence generates a difference in electrochemical potential of protons across the inner mitochondrial membrane. This potential difference drives the synthesis of adenosine triphosphate (ATP), which is the universal energy carrier within all biological cells. rnrnThe goal of the present work is to contribute to a better understanding of the functional mechanism of CcO by using time-resolved surface enhanced resonance Raman spectroscopy (TR-SERRS). Despite intensive research effort within the last decades, the functional mechanism of CcO is still subject to controversial discussions. It was the primary goal of this dissertation to initiate electron transfer to the redox centers CuA, heme a, heme a3 and CuB electrochemically and to observe the corresponding redox transitions in-situ with a focus on the two heme structures by using SERRS. A measuring cell was developed, which allowed combination of electrochemical excitation with Raman spectroscopy for the purpose of performing the accordant measurements. Cytochrome c was used as a benchmark system to test the new measuring cell and to prove the feasibility of appropriate Raman measurements. In contrast to CcO the heme protein cc contains only a single heme structure. Nevertheless, characteristic Raman bands of the hemes can be observed for both proteins.rnrnIn order to investigate CcO it was immobilized on top of a silver substrate and embedded into an artificial membrane. The catalytic activity of CcO and therefore the complete functional capability of the enzyme within the biomimetic membrane architecture was verified using cyclic voltammetry. Raman spectroscopy was performed using a special nano-structured silver surface, which was developed within the scope of the present work. This new substrate combined two fundamental properties. It facilitated the formation of a protein tethered bilayer lipid membrane (ptBLM) and it allowed obtaining Raman spectra with sufficient high signal-to-noise ratios.rnSpectro-electrochemical investigations showed that at open circuit potential the enzyme exists in a mixed-valence state, with heme a and and heme a3 in the reduced and oxidized state, respectively. This was considered as an intermediate state between the non-activated and the fully activated state of CcO. Time-resolved SERRS measurements revealed that a hampered electron transfer to the redox center heme a3 characterizes this intermediate state.rn

Investigation of the electric field enhanced PNA-DNA hybridization on Au surface by using surface plasmon field-enhanced fluorescence spectroscopy (SPFS)

Recently, the surface plasmon field-enhanced fluorescence spectroscopy (SPFS) was developed as a kinetic analysis and a detection method with dual- monitoring of the change of reflectivity and fluorescence signal for the interfacial phenomenon. A fundamental study of PNA and DNA interaction at the surface using surface plasmon fluorescence spectroscopy (SPFS) will be investigated in studies. Furthermore, several specific conditions to influence on PNA/DNA hybridization and affinity efficiency by monitoring reflective index changes and fluorescence variation at the same time will be considered. In order to identify the affinity degree of PNA/DNA hybridizaiton at the surface, the association constant (kon) and the dissociation constant (koff) will be obtained by titration experiment of various concentration of target DNA and kinetic investigation. In addition, for more enhancing the hybridization efficiency of PNA/DNA, a study of polarized electric field enhancement system will be introduced and performed in detail. DNA is well-known polyelectrolytes with naturally negative charged molecules in its structure. With polarized electrical treatment, applying DC field to the metal surface, which PNA probe would be immobilized at, negatively charged DNA molecules can be attracted by electromagnetic attraction force and manipulated to the close the surface area, and have more possibility to hybridize with probe PNA molecules by hydrogen bonding each corresponding base sequence. There are several major factors can be influenced on the hybridization efficiency.

Plasmon-enhanced dynamic light scattering of gold nanorods in bulk and near a wall

Transportprozesse von anisotropen metallischen Nanopartikeln wie zum Beispiel Gold-Nanostäbchen in komplexen Flüssigkeiten und/oder begrenzten Geometrien spielen eine bedeutende Rolle in einer Vielzahl von biomedizinischen und industriellen Anwendungen. Ein Weg zu einem tiefen, grundlegenden Verständnis von Transportmechanismen ist die Verwendung zweier leistungsstarker Methoden - dynamischer Lichtstreuung (DLS) und resonanzverstärkter Lichtstreuung (REDLS) in der Nähe einer Grenzfläche. In dieser Arbeit wurden nanomolare Suspensionen von Gold-Nanostäbchen, stabilisiert mit Cetyltrimethylammoniumbromid (CTAB), mit DLS sowie in der Nähe einer Grenzfläche mit REDLS untersucht. Mit DLS wurde eine wellenlängenabhängige Verstärkung der anisotropen Streuung beobachtet, welche sich durch die Anregung von longitudinaler Oberflächenplasmonenresonanz ergibt. Die hohe Streuintensität nahe der longitudinalen Oberflächenplasmonenresonanzfrequenz für Stäbchen, welche parallel zum anregenden optischen Feld liegen, erlaubte die Auflösung der translationalen Anisotropie in einem isotropen Medium. Diese wellenlängenabhängige anisotrope Lichtstreuung ermöglicht neue Anwendungen wie etwa die Untersuchung der Dynamik einzelner Partikel in komplexen Umgebungen mittels depolarisierter dynamischer Lichtstreuung. In der Nähe einer Grenzfläche wurde eine starke Verlangsamung der translationalen Diffusion beobachtet. Hingegen zeigte sich für die Rotation zwar eine ausgeprägte aber weniger starke Verlangsamung. Um den möglichen Einfluss von Ladung auf der festen Grenzfläche zu untersuchen, wurde das Metall mit elektrisch neutralem Polymethylmethacrylat (PMMA) beschichtet. In einem weiteren Ansatz wurde das CTAB in der Gold-Nanostäbchen Lösung durch das kovalent gebundene 16-Mercaptohexadecyltrimethylammoniumbromid (MTAB) ersetzt. Daraus ergab sich eine deutlich geringere Verlangsamung.

New enhancement strategies for plasmon-enhanced fluorescence biosensors

This thesis investigates metallic nanostructures exhibiting surface plasmon resonance for the amplification of fluorescence signal in sandwich immunoassays. In this approach, an analyte is captured by an antibody immobilized on a plasmonic structure and detected by a subsequently bound fluorophore labeled detection antibody. The highly confined field of surface plasmons originates from collective charge oscillations which are associated with high electromagnetic field enhancements at the metal surface and allow for greatly increased fluorescence signal from the attached fluorophores. This feature allows for improving the signal-to-noise ratio in fluorescence measurements and thus advancing the sensitivity of the sensor platform. In particular, the thesis presents two plasmonic nanostructures that amplify fluorescence signal in devices that rely on epifluorescence geometry, in which the fluorophore absorbs and emits light from the same direction perpendicular to the substrate surface.rnThe first is a crossed relief gold grating that supports propagating surface plasmon polaritons (SPPs) and second, gold nanoparticles embedded in refractive index symmetric environment exhibiting collective localized surface plasmons (cLSPs). Finite-difference time-domain simulations are performed in order to design structures for the optimum amplification of established Cy5 and Alexa Fluor 647 fluorophore labels with the absorption and emission wavelengths in the red region of spectrum. The design takes into account combined effect of surface plasmon-enhanced excitation rate, directional surface plasmon-driven emission and modified quantum yield for characteristic distances in immunoassays. Homebuilt optical instruments are developed for the experimental observation of the surface plasmon mode spectrum, measurements of the angular distribution of surface plasmon-coupled fluorescence light and a setup mimicking commercial fluorescence reading systems in epifluorescence geometry.rnCrossed relief grating structures are prepared by interference lithography and multiple copies are made by UV nanoimprint lithography. The fabricated crossed diffraction gratings were utilized for sandwich immunoassay-based detection of the clinically relevant inflammation marker interleukin 6 (IL-6). The enhancement factor of the crossed grating reached EF=100 when compared to a flat gold substrate. This result is comparable to the highest reported enhancements to date, for fluorophores with relatively high intrinsic quantum yield. The measured enhancement factor excellently agrees with the predictions of the simulations and the mechanisms of the enhancement are explained in detail. Main contributions were the high electric field intensity enhancement (30-fold increase) and the directional fluorescence emission at (4-fold increase) compared to a flat gold substrate.rnCollective localized surface plasmons (cLSPs) hold potential for even stronger fluorescence enhancement of EF=1000, due to higher electric field intensity confinement. cLSPs are established by diffractive coupling of the localized surface plasmon resonance (LSPR) of metallic nanoparticles and result in a narrow resonance. Due to the narrow resonance, it is hard to overlap the cLSPs mode with the absorption and emission bands of the used fluorophore, simultaneously. Therefore, a novel two resonance structure that supports SPP and cLSP modes was proposed. It consists of a 2D array of cylindrical gold nanoparticles above a low refractive index polymer and a silver film. A structure that supports the proposed SPP and cLSP modes was prepared by employing laser interference lithography and the measured mode spectrum was compared to simulation results.rn

Host-pathogen interactions of secreted and surface Staphylococcus aureus factors

Staphylococcus aureus is an opportunistic bacterial pathogen that can infect humans and other species. It utilizes an arsenal of virulence factors to cause disease, including secreted and cell wall anchored factors. Secreted toxins attack host cells, and pore-forming toxins destroy target cells by causing cell lysis. S. aureus uses cell-surface adhesins to attach to host molecules thereby facilitating host colonization. The Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) are a family of cell-wall anchored proteins that target molecules like fibronectin and fibrinogen. The Serine-aspartate repeat (Sdr) proteins are a subset of staphylococcal MSCRAMMs that share similar domain organization. Interestingly, the amino-terminus, is composed of three immunoglobulin-folded subdomains (N1, N2, and N3) that contain ligand-binding activity. Clumping factors A and B (ClfA and ClfB) and SdrG are Sdr proteins that bind to fibrinogen (Fg), a large, plasma glycoprotein that is activated during the clotting cascade to form fibrin. In addition to recognizing fibrinogen, ClfA and ClfB can bind to other host ligands. Analysis of S. aureus strains that cause osteomyelitis led to the discovery of the bone-sialoprotein-binding protein (Bbp), an Sdr protein. Because several MSCRAMMs target more than one molecule, I hypothesized that Bbp may recognize other host proteins. A ligand screen revealed that the recombinant construct BbpN2N3 specifically recognizes human Fg. Surface plasmon resonance was used to determine the affinity of BbpN2N3 for Fg, and a dissociation constant of 540 nM was determined. Binding experiments performed with recombinant Fg chains were used to map the binding of BbpN2N3 to the Fg Aalpha chain. Additionally, Bbp expressed on the surface of Lactococcus lactis and S. aureus Newman bald mediated attachment of these bacteria to Fg Aalpha. To further characterize the interaction between the two proteins, isothermal titration calorimetry and inhibition assays were conducted with synthetic Fg Aalpha peptides. To determine the physiological implications of Bbp binding to Fg, the effect of Bbp on fibrinogen clotting was studied. Results show that Bbp binding to Fg inhibits the formation of fibrin. The consequences of this interaction are currently under investigation. Together, these data demonstrate that human Fg is a novel ligand for Bbp. This study indicates that the MSCRAMM Bbp may aid in staphylococcal attachment by targeting both an extracellular matrix and a blood plasma protein. The implications of these novel findings are discussed.

Self-organized colloidal quantum dots and metal nanoparticles for plasmon-enhanced intermediate-band solar cells

A colloidal deposition technique is presented to construct long-range ordered hybrid arrays of self-assembled quantum dots and metal nanoparticles. Quantum dots are promising for novel opto-electronic devices but, in most cases, their optical transitions of interest lack sufficient light absorption to provide a significant impact in their implementation. A potential solution is to couple the dots with localized plasmons in metal nanoparticles. The extreme confinement of light in the near-field produced by the nanoparticles can potentially boost the absorption in the quantum dots by up to two orders of magnitude. In this work, light extinction measurements are employed to probe the plasmon resonance of spherical gold nanoparticles in lead sulfide colloidal quantum dots and amorphous silicon thin-films. Mie theory computations are used to analyze the experimental results and determine the absorption enhancement that can be generated by the highly intense near-field produced in the vicinity of the gold nanoparticles at their surface plasmon resonance. The results presented here are of interest for the development of plasmon-enhanced colloidal nanostructured photovoltaic materials, such as colloidal quantum dot intermediate-band solar cells.

Thermally tunable polarization by nanoparticle plasmonic resonance in photonic crystal fibers

A photonic crystal fiber selectively filled with silver nanoparticles dispersed in polydimethylsiloxane has been numerically studied via finite elements analysis. These nanoparticles possess a localized surface plasmon resonance in the visible region which depends on the refractive index of the surrounding medium. The refractive index of polydimethylsiloxane can be thermally tuned leading to the design of polarization tunable filters. Filters found with this setup show anisotropic attenuation of the x-polarization fundamental mode around ?x = 1200dB/cm remarkably higher than the y-polarization mode. Moreover, high fiber birefringence and birefringence reversal is observed in the spectral region of the plasmon.

Determination of the lifetime and force dependence of interactions of single bonds between surface-attached CD2 and CD48 adhesion molecules

We studied single molecular interactions between surface-attached rat CD2, a T-lymphocyte adhesion receptor, and CD48, a CD2 ligand found on antigen-presenting cells. Spherical particles were coated with decreasing densities of CD48–CD4 chimeric molecules then driven along CD2-derivatized glass surfaces under a low hydrodynamic shear rate. Particles exhibited multiple arrests of varying duration. By analyzing the dependence of arrest frequency and duration on the surface density of CD48 sites, it was concluded that (i) arrests were generated by single molecular bonds and (ii) the initial bond dissociation rate was about 7.8 s−1. The force exerted on bonds was increased from about 11 to 22 pN; the detachment rate exhibited a twofold increase. These results agree with and extend studies on the CD2–CD48 interaction by surface plasmon resonance technology, which yielded an affinity constant of ≈104 M−1 and a dissociation rate of ≥6 s−1. It is concluded that the flow chamber technology can be an useful complement to atomic force microscopy for studying interactions between isolated biomolecules, with a resolution of about 20 ms and sensitivity of a few piconewtons. Further, this technology might be extended to actual cells.

Analysis of the interaction of ZAP-70 and syk protein-tyrosine kinases with the T-cell antigen receptor by plasmon resonance.

Tyrosine phosphorylation of a 17-amino acid immunoreceptor tyrosine-based activation motif (ITAM), conserved in each of the signaling subunits of the T-cell antigen receptor (TCR), mediates the recruitment of ZAP-70 and syk protein-tyrosine kinases (PTKs) to the activated receptor. The interaction between the two tandemly arranged Src-homology 2 (SH2) domains of this family of PTKs and each of the phosphotyrosine-containing ITAMs was examined by real-time measurements of kinetic parameters. The association rate and equilibrium binding constants for the ZAP-70 and syk SH2 domains were determined for the CD3 epsilon ITAM. Both PTKs bound with ka and Kd values of 5 x 10(6) M-1.sec-1 and approximately 25 nM, respectively. Bindings to the other TCR ITAMs (zeta 1, zeta 2, gamma, and delta ITAMs) were comparable, although the zeta 3 ITAM bound approximately 2.5-fold less well. Studies of the affinity of a single functional SH2 domain of ZAP-70 provided evidence for the cooperative nature of binding of the dual SH2 domains. Mutation of either single SH2 domain decreased the Kd by > 100-fold. Finally, the critical features of the ITAM for syk binding were found to be similar to those required for ZAP-70 binding. These data provide insight into the mechanism by which the multisubunit TCR interacts with downstream effector molecules.

Generation of infrared surface plasmon resonances with high refractive index sensitivity utilizing tilted fiber Bragg gratings

We demonstrate the use of tilted fiber gratings to assist the generation of localized infrared surface plasmons with short propagation lengths and a sensitivity of d lambda/dn = 3365 nm in the aqueous index regime. It was also found that the resonances could be spectrally tuned over 1000 nm at the same spatial region with high coupling efficiency (in excess of 25 dB) by altering the polarization of the light illuminating the device.

Aptamer-based surface plasmon sensor for thrombin detection

A series of surface plasmonic fibre devices were fabricated using multiple coatings deposited on a lapped section of a single mode fibre and post-fabrication UV laser irradiation processing with a phase mask, producing a surface relief grating structure. These devices showed high spectral sensitivity in the aqueous index regime ranging up to 4000 nm/RIU for wavelength and 800 dB/RIU for intensity. The devices were then coated with human thrombin binding aptamer. Several concentrations of thrombin in buffer solution were made, ranging from 1nM to 1µM. All the concentrations were detectable by the devices demonstrating that sub-nM concentrations may be monitored.

Generation of infrared surface plasmon resonances with high refractive index sensitivity utilizing tilted fiber Bragg gratings

We demonstrate the use of tilted fiber gratings to assist the generation of localized infrared surface Plasmons with short propagation lengths and a sensitivity of dλ/dn = 3365 nm in the aqueous index regime. It was also found that the resonances could be spectrally tuned over 1000 nm at the same spatial region with high coupling efficiency (in excess of 25 dB) by altering the polarization of the light illuminating the device. © 2007 Optical Society of America.

Photonic gas sensors exploiting directly the optical properties of hybrid carbon nanotube localized surface plasmon structures

We investigate the modification of the optical properties of carbon nanotubes (CNTs) resulting from a chemical reaction triggered by the presence of a specific compound (gaseous carbon dioxide (CO2)) and show this mechanism has important consequences for chemical sensing. CNTs have attracted significant research interest because they can be functionalized for a particular chemical, yielding a specific physical response which suggests many potential applications in the fields of nanotechnology and sensing. So far, however, utilizing their optical properties for this purpose has proven to be challenging. We demonstrate the use of localized surface plasmons generated on a nanostructured thin film, resembling a large array of nano-wires, to detect changes in the optical properties of the CNTs. Chemical selectivity is demonstrated using CO2 in gaseous form at room temperature. The demonstrated methodology results additionally in a new, electrically passive, optical sensing configuration that opens up the possibilities of using CNTs as sensors in hazardous/explosive environments.