New biosensor applications of surface plasmon and hydrogel optical waveguide spectroscopy

Rapid and sensitive detection of chemical and biological analytes becomes increasingly important in areas such as medical diagnostics, food control and environmental monitoring. Optical biosensors based on surface plasmon resonance (SPR) and optical waveguide spectroscopy have been extensively pushed forward in these fields. In this study, we combine SPR, surface plasmon-enhanced fluorescence spectroscopy (SPFS) and optical waveguide spectroscopy with hydrogel thin film for highly sensitive detection of molecular analytes.rnrnA novel biosensor based on SPFS which was advanced through the excitation of long range surface plasmons (LRSPs) is reported in this study. LRSPs are special surface plasmon waves propagating along thin metal films with orders of magnitude higher electromagnetic field intensity and lower damping than conventional SPs. Therefore, their excitation on the sensor surface provides further increased fluorescence signal. An inhibition immunoassay based on LRSP-enhanced fluorescence spectroscopy (LRSP-FS) was developed for the detection of aflatoxin M1 (AFM1) in milk. The biosensor allowed for the detection of AFM1 in milk at concentrations as low as 0.6 pg mL-1, which is about two orders of magnitude lower than the maximum AFM1 residue level in milk stipulated by the European Commission legislation.rnrnIn addition, LRSPs probe the medium adjacent to the metallic surface with more extended evanescent field than regular SPs. Therefore, three-dimensional binding matrices with up to micrometer thickness have been proposed for the immobilization of biomolecular recognition elements with large surface density that allows to exploit the whole evanescent field of LRSP. A photocrosslinkable carboxymethyl dextran (PCDM) hydrogel thin film is used as a binding matrix, and it is applied for the detection of free prostate specific antigen (f-PSA) based on the LRSP-FS and sandwich immunoassay. We show that this approach allows for the detection of f-PSA at low femto-molar range, which is approximately four orders of magnitude lower than that for direct detection of f-PSA based on the monitoring of binding-induced refractive index changes.rnrnHowever, a three dimensional hydrogel binding matrix with micrometer thickness can also serve as an optical waveguide. Based on the measurement of binding-induced refractive index changes, a hydrogel optical waveguide spectroscopy (HOWS) is reported for a label-free biosensor. This biosensor is implemented by using a SPR optical setup in which a carboxylated poly(N-isoproprylacrylamide) (PNIPAAm) hydrogel film is attached on a metallic surface and modified by protein catcher molecules. Compared to regular SPR biosensor with thiol self-assembled monolayer (SAM), HOWS provides an order of magnitude improved resolution in the refractive index measurements and enlarged binding capacity owing to its low damping and large swelling ratio, respectively. A model immunoassay experiment revealed that HOWS allowed detection of IgG molecules with a 10 pM limit of detection (LOD) that was five-fold lower than that achieved for SPR with thiol SAM. For the high capacity hydrogel matrix, the affinity binding was mass transport limited.rnrnThe mass transport of target molecules to the sensor surface can play as critical a role as the chemical reaction itself. In order to overcome the diffusion-limited mass transfer, magnetic iron oxide nanoparticles were employed. The magnetic nanoparticles (MNPs) can serve both as labels providing enhancement of the refractive index changes, and “vehicles” for rapidly delivering the analytes from sample solution to an SPR sensor surface with a gradient magnetic field. A model sandwich assay for the detection of β human chorionic gonadotropin (βhCG) has been utilized on a gold sensor surface with metallic diffraction grating structure supporting the excitation of SPs. Various detection formats including a) direct detection, b) sandwich assay, c) MNPs immunoassay without and d) with applied magnetic field were compared. The results show that the highly-sensitive MNPs immunoassay improves the LOD on the detection of βhCG by a factor of 5 orders of magnitude with respect to the direct detection.rn

Direct electron transfer to cytochrome c oxidase investigated by electrochemistry and time-resolved surface-enhanced infrared absorption spectroscopy

Cytochrom c Oxidase (CcO), der Komplex IV der Atmungskette, ist eine der Häm-Kupfer enthaltenden Oxidasen und hat eine wichtige Funktion im Zellmetabolismus. Das Enzym enthält vier prosthetische Gruppen und befindet sich in der inneren Membran von Mitochondrien und in der Zellmembran einiger aerober Bakterien. Die CcO katalysiert den Elektronentransfer (ET) von Cytochrom c zu O2, wobei die eigentliche Reaktion am binuklearen Zentrum (CuB-Häm a3) erfolgt. Bei der Reduktion von O2 zu zwei H2O werden vier Protonen verbraucht. Zudem werden vier Protonen über die Membran transportiert, wodurch eine elektrochemische Potentialdifferenz dieser Ionen zwischen Matrix und Intermembranphase entsteht. Trotz ihrer Wichtigkeit sind Membranproteine wie die CcO noch wenig untersucht, weshalb auch der Mechanismus der Atmungskette noch nicht vollständig aufgeklärt ist. Das Ziel dieser Arbeit ist, einen Beitrag zum Verständnis der Funktion der CcO zu leisten. Hierzu wurde die CcO aus Rhodobacter sphaeroides über einen His-Anker, der am C-Terminus der Untereinheit II angebracht wurde, an eine funktionalisierte Metallelektrode in definierter Orientierung gebunden. Der erste Elektronenakzeptor, das CuA, liegt dabei am nächsten zur Metalloberfläche. Dann wurde eine Doppelschicht aus Lipiden insitu zwischen die gebundenen Proteine eingefügt, was zur sog. proteingebundenen Lipid-Doppelschicht Membran (ptBLM) führt. Dabei musste die optimale Oberflächenkonzentration der gebundenen Proteine herausgefunden werden. Elektrochemische Impedanzspektroskopie(EIS), Oberflächenplasmonenresonanzspektroskopie (SPR) und zyklische Voltammetrie (CV) wurden angewandt um die Aktivität der CcO als Funktion der Packungsdichte zu charakterisieren. Der Hauptteil der Arbeit betrifft die Untersuchung des direkten ET zur CcO unter anaeroben Bedingungen. Die Kombination aus zeitaufgelöster oberflächenverstärkter Infrarot-Absorptionsspektroskopie (tr-SEIRAS) und Elektrochemie hat sich dafür als besonders geeignet erwiesen. In einer ersten Studie wurde der ET mit Hilfe von fast scan CV untersucht, wobei CVs von nicht-aktivierter sowie aktivierter CcO mit verschiedenen Vorschubgeschwindigkeiten gemessen wurden. Die aktivierte Form wurde nach dem katalytischen Umsatz des Proteins in Anwesenheit von O2 erhalten. Ein vier-ET-modell wurde entwickelt um die CVs zu analysieren. Die Methode erlaubt zwischen dem Mechanismus des sequentiellen und des unabhängigen ET zu den vier Zentren CuA, Häm a, Häm a3 und CuB zu unterscheiden. Zudem lassen sich die Standardredoxpotentiale und die kinetischen Koeffizienten des ET bestimmen. In einer zweiten Studie wurde tr-SEIRAS im step scan Modus angewandt. Dafür wurden Rechteckpulse an die CcO angelegt und SEIRAS im ART-Modus verwendet um Spektren bei definierten Zeitscheiben aufzunehmen. Aus diesen Spektren wurden einzelne Banden isoliert, die Veränderungen von Vibrationsmoden der Aminosäuren und Peptidgruppen in Abhängigkeit des Redoxzustands der Zentren zeigen. Aufgrund von Zuordnungen aus der Literatur, die durch potentiometrische Titration der CcO ermittelt wurden, konnten die Banden versuchsweise den Redoxzentren zugeordnet werden. Die Bandenflächen gegen die Zeit aufgetragen geben dann die Redox-Kinetik der Zentren wieder und wurden wiederum mit dem vier-ET-Modell ausgewertet. Die Ergebnisse beider Studien erlauben die Schlussfolgerung, dass der ET zur CcO in einer ptBLM mit größter Wahrscheinlichkeit dem sequentiellen Mechanismus folgt, was dem natürlichen ET von Cytochrom c zur CcO entspricht.

On-surface chemical reactions on an insulating substrate

Diese Arbeit beschreibt zum ersten Mal die kovalente Verknüpfung organischer Moleküle auf einer Isolatoroberfläche, motiviert im Hinblick auf die Nutzung der Synthesemethode für die molekulare Elektronik und verwandte Anwendungen. Durch die Verwendung der Nichtkontakt-Rasterkraftmikroskopie und der Kelvinprobe-Mikroskopie bei Raumtemperatur wurden grundlegende molekulare Prozesse der Wechselwirkungen zwischen Molekülen und der Calcit(10.4) Oberfläche sowie die chemische Reaktivität der Moleküle auf der Oberfläche analysiert. Das Zusammenspiel zwischen intermolekularen und Molekül-Oberfläche Wechselwirkungen zeigt sich für Biphenyl-4,4'-dicarbonsäure (BPDCA) durch die Koexistenz zweier unterschiedlicher molekularer Strukturen, die einen Einblick in die treibenden Kräfte der molekularen Selbstorganisation bieten. Die sehr ausgeprägte Reihenstruktur basiert auf der optimalen geometrischen Struktur der BPDCA Moleküle zu den Abmessungen des Substrats, während die zweite Struktur durch Wasserstoffbrücken zwischen den Molekülen gekennzeichnet ist. Der Deprotonierungsvorgang von 2,5-Dihydroxybenzoesäure (DHBA)-Molekülen auf Calcit wird bei Zimmertemperatur gezeigt. Zwei Phasen werden beobachtet, die nach Aufbringen der Moleküle koexistieren. Mit der Zeit geht eine bulk-ähnliche Phase in eine stabile, dicht gepackte Phase über. Der Übergang wird durch Betrachtung des Protonierungszustands der Moleküle erklärt. Die bulk-ähnliche Phase benötigt Wasserstoffbrückbindungen zur Strukturbildung. Werden die Moleküle deprotoniert, so wird die resultierende dicht gepackte Phase durch die elektrostatische Wechselwirkung der deprotonierten Carboxylatgruppen mit den Oberflächen-Calciumkationen stabilisiert. 4-Iodbenzoesäure (IBA)-Moleküle bilden auf Calcit nur Inseln an Stufenkanten, was auf die schwache Molekül-Oberflächen-Wechselwirkung zurückzuführen ist. Für einen stärkeren Einfluss des Substrats durchlaufen die Moleküle einen kontrollierten Übergangsschritt vom protonierten zum deprotonierten Zustand. Im deprotonierten Zustand nehmen die Moleküle eine wohldefinierte Adsorptionsposition auf dem Substrat ein. Die deprotonierte Säuregruppe wird ausgenutzt, um die Desorption der halogensubstituierten Benzoesäure-Moleküle bei der thermischer Aktivierung für die Vernetzungsreaktion zu vermeiden. Darüber hinaus wird die Carboxylatgruppe als starker Elektronendonor verwendet um die Phenyl-Halogen-Bindung zu schwächen und somit die homolytische Spaltung dieser Bindung auch bei moderaten Temperaturen zu ermöglichen. Diesem Konzept folgend ist die erste erfolgreiche kovalente Verknüpfung von 2,5-Diiod-benzoesäure, 2,5-Dichlorbenzoesäure, 3,5-Diiod Salicylsäure und 4-Iod-benzoesäure zu durchkonjugierten molekularen Drähten, Zick-Zack-Strukturen sowie Dimere gezeigt durch Ausnutzen von unterschiedlichen Substitutionsposition sowie Ändern der Anzahl der substituierten Halogenatome. Aufbauend auf diesem Erfolg, wird eine zweistufige Vernetzungsreaktion vorgestellt. Zum Induzieren der ortsspezifischen und sequentiellen kovalenten Verknüpfung wird ein Ausgangsmolekül gewählt, das sowohl eine Bromphenyl als auch eine Chlorphenyl Gruppe mit unterschiedlichen Dissoziationsenergien für die homolytische Spaltung besitzt. Die Reaktionsstellen und sequentielle Reihenfolge für die Reaktion sind somit in der molekularen Struktur einkodiert und bisher unerreichte Reaktionspfade können mithilfe der kovalente Verknüpfung organischer Moleküle auf einer Isolatoroberfläche beschritten werden.

Stabilizing molecular self-assemblies via on-surface reactions

Für die Realisierung zukünftiger Technologien, wie z.B. molekulare Elektronik, werden Strategien benötigt, um funktionale Strukturen direkt auf Oberflächen zu erzeugen. Für die Bewältigung dieser Aufgabe ist die molekulare Selbstanordnung ein äußerst vielversprechender Bottom-up-Ansatz. Hierbei ist eine der größten Herausforderungen das Zusammenspiel aus intramolekularer Wechselwirkung und der Wechselwirkung zwischen Substrat und Molekülen in ein Gleichgewicht zu bringen. Da jedoch die wirkenden Kräfte der molekularen Selbstanordnung ausschließlich reversibler Natur sind, ist eine langfristige Stabilität fragwürdig. Somit ist die kovalente Verknüpfung der gebildeten Strukturen durch Reaktionen direkt auf der Oberfläche unerlässlich, um die Stabilität der Strukturen weiter zu erhöhen. Hierzu stellt die vorliegende Arbeit eine ausführliche Studie zu molekularer Selbstanordnung und der zielgerichteten Modifikation ebensolcher Strukturen dar. Durch den Einsatz von hochauflösender Rasterkraftmikroskopie im Ultrahochvakuum, welche es erlaubt einzelne Moleküle auf Nichtleitern abzubilden, wurde der maßgebliche Einfluss von Ankerfunktionalitäten auf den Prozess der molekularen Selbstanordnung gezeigt. Des Weiteren konnte die Stabilität der selbst angeordneten Strukturen durch neue Oberflächenreaktionskonzepte entschieden verbessert werden. Der Einfluss von Ankerfunktionen, die elektrostatische Wechselwirkung zwischen Molekül und Substrat vermitteln, auf den Strukturbildungsprozess der molekularen Selbstanordnung wird eingehend durch den Vergleich eines aromatischen Moleküls und seines vierfach chlorierten Derivates gezeigt. Für diese beiden Moleküle wurde ein deutlich unterschiedliches Verhalten der Selbstanordnung beobachtet. Es wird gezeigt, dass die Fähigkeit zur Bildung selbst angeordneter, stabiler Inseln entscheidend durch die Substituenten und die Abmessungen des Moleküls beeinflusst wird. Auch wird in dieser Arbeit die erste photochemische Reaktion organischer Moleküle auf einem Isolator gezeigt. Qualitative und quantitative Ergebnisse liefern ein detailliertes Bild darüber, wie die Abmessungen des Substratgitters die Richtung der Reaktion gezielt beeinflussen. Des Weiteren wird ein allgemeines Konzept zur selektiven Stabilisierung selbstangeordneter Molekülstrukturen durch den kontrollierten Transfer von Elektronen präsentiert. Durch die gezielte Steuerung der Menge an Dotierungsatomen wird die Desorptionstemperatur der molekularen Inseln signifikant erhöht und das Desorptionsverhalten der Inseln entschieden verändert. Diese Arbeit präsentiert somit erfolgreich durchgeführte Strategien um den Prozess der molekularen Selbstanordnung zu steuern, sowie entscheidende Mechanismen um die Stabilisierung und Modifizierung von selbst angeordneten Strukturen zu gewährleisten.

Synthesis and Surface Modification of CdSe and CdS Quantum Dots Exhibiting High Quantum Yield

The thesis investigates the effect of surface treatment with various reducing and oxidizing agents on the quantum yield (QY) of CdSe and CdS quantum dots (QDs). The QDs, as synthesized by the organometallic method, contained defect sites on their surface that trapped photons and prevented their radiative recombination, therefore resulting in adecreased QY. To passivate these defect sites and enhance the QY, the QDs were treated with various reducing and oxidizing agents, including: sodium borohydride (NaBH4), calcium hydride (CaH2), hydrazine (N2H4), benzoyl peroxide (C14H10O4), and tert-butylhydroperoxide (C4H10O2). It was hypothesized that the reducing/oxidizing agents reduced the ligands on the QD surface, causing them to detach, thereby allowing oxygen from atmospheric air to bind to the exposed cadmium. This cadmium oxdide (CdO) layeraround the QD surface satisfied the defect sites and resulted in an increased QY. To correlate what effect the reducing and oxidizing agents were having on the optical properties of the QDs, we investigated these treatments on the following factors:chalcogenide (Se vs. S), ligand (oleylamine vs. OA), coordinating solvent (ODE vs.TOA), and dispersant solvent (chloroform vs. toluene) on the overall optical properties of the QDs. The QY of each sample was calculated before and after the various surface treatments from ultra-violet visible spectroscopy (UV-Vis) and fluorescence spectroscopy data to determine if the treatment was successful.From our results, we found that sodium borohydride was the most effective surface treatment, with 10 of the 12 treatments resulting in an increased QY. Hydrazine, on the other hand, was the least effective treatments, as it quenched the QD fluorescence in every case. From these observations, we hypothesize that the effectiveness of the QD surface treatments was dependent on reaction rate. More specifically, when the surface treatment reaction happened too quickly, we hypothesize that the QDs began to aggregate, resulting in a quenched fluorescence. Furthermore, we believe that the reactionrate is dependent on concentration of the reducing/oxidizing agents, solubility of the agents in each solvent, and reactivity of the agents with water. The quantum yield of the QDs can therefore be maximized by slowing the reaction rate of each surface treatment toa rate that allows for the proper passivation of defect sites.

cDNA cloning and expression of a novel cell surface-expressed \b heparan sulfate/heparin \b interacting \b protein (HIP) from human cell lines and functional studies of a synthetic HIP peptide

Heparan sulfate proteoglycans and their corresponding binding sites have been suggested to play an important role during the initial attachment of blastocysts to uterine epithelium and human trophoblastic cell lines to uterine epithelial cell lines. Previous studies on RL95 cells, a human uterine epithelial cell line, characterized a single class of cell surface heparin/heparan sulfate (HP/HS)-binding sites. Three major HP/HS-binding peptide fragments were isolated from RL95 cell surfaces by tryptic digestion and partial amino-terminal amino acid sequence from each peptide fragment was obtained. In the current study, using the approaches of reverse transcription-polymerase chain reaction and cDNA library screening, a novel cell surface $\rm\underline{H}$P/HS $\rm\underline{i}$nteracting $\rm\underline{p}$rotein (HIP) has been isolated from RL95 cells. The full-length cDNA of HIP encodes a protein of 259 amino acids with a calculated molecular weight of 17,754 Da and pI of 11.75. Transfection of HIP cDNA into NIH-3T3 cells demonstrated cell surface expression and a size similar to that of HIP expressed by human cells. Predicted amino acid sequence indicates that HIP lacks a membrane spanning region and has no consensus sites for glycosylation. Northern blot analysis detected a single transcript of 1.3 kb in both total RNA and poly(A$\sp+$) RNA. Examination of human cell lines and normal tissues using both Northern blot and Western blot analysis revealed that HIP is differentially expressed in a variety of human cell lines and normal tissues, but absent in some cell lines examined. HIP has about 80% homology, at the level of both mRNA and protein, to a rodent protein, designated as ribosomal protein L29. Thus, members of the L29 family may be displayed on cell surfaces where they participate in HP/HS binding events. Studies on a synthetic peptide derived from HIP demonstrate that HIP peptide binds HS/HP with high selectivity and has high affinity (Kd = 10 nM) for a subset of polysaccharides found in commercial HIP preparations. Moreover, HIP peptide also binds certain forms of cell surface, but not secreted or intracellular. HS expressed by RL95 and JAR cells. This peptide supports the attachment of several human trophoblastic cell lines and a variety of mammalian adherent cell lines in a HS-dependent fashion. Furthermore, studies on the subset of HP specifically recognized by HIP peptide indicate that this high-affinity HP (HA-HP) has a larger median MW and a greater negative charge density than bulk HP. The minimum size of oligosaccharide required to bind to HIP peptide with high affinity is a septa- or octasaccharide. HA-HP also quantitatively binds to antithrombin-III (AT-III) with high affinity, indicating that HIP peptide and AT-III may recognize the same or similar oligosaccharide structure(s). Furthermore, HIP peptide antagonizes HP action and promotes blood coagulation in both factor Xa- and thrombin-dependent assays. Finally, HA-HP recognized by HP peptide is highly enriched with anticoagulant activity relative to bulk HP. Collectively, these results demonstrate that HIP may play a role in the HP/HS-involved cell-cell and cell-matrix interactions and recognizes a motif in HP similar or identical to that recognized by AT-III and therefore, may modulate blood coagulation. ^

Probing the Electrocatalytic Oxygen Reduction Reaction Reactivity of Immobilized Multicopper Oxidase CueO

The bioelectrocatalytic (oxygen reduction reaction, ORR) properties of the multicopper oxidase CueO immobilized on gold electrodes were investigated. Macroscopic electrochemical techniques were combined with in situ scanning tunneling microscopy (STM) and surface-enhanced Raman spectroscopy at the ensemble and at the single-molecule level. Self-assembled monolayer of mercaptopropionic acid, cysteamine, and p-aminothiophenol were chosen as redox mediators. The highest ORR activity was observed for the protein attached to amino-terminated adlayers. In situ STM experiments revealed that the presence of oxygen causes distinct structure and electronic changes in the metallic centers of the enzyme, which determine the rate of intramolecular electron transfer and, consequently, affect the rate of electron tunneling through the protein. Complementary Raman spectroscopy experiments provided access for monitoring structural changes in the redox state of the type 1 copper center of the immobilized enzyme during the CueO-catalyzed oxygen reduction cycle. These results unequivocally demonstrate the existence of a direct electronic communication between the electrode substrate and the type 1 copper center.

Comparison of (31)P saturation and inversion magnetization transfer in human liver and skeletal muscle using a clinical MR system and surface coils.

(31)P MRS magnetization transfer ((31)P-MT) experiments allow the estimation of exchange rates of biochemical reactions, such as the creatine kinase equilibrium and adenosine triphosphate (ATP) synthesis. Although various (31)P-MT methods have been successfully used on isolated organs or animals, their application on humans in clinical scanners poses specific challenges. This study compared two major (31)P-MT methods on a clinical MR system using heteronuclear surface coils. Although saturation transfer (ST) is the most commonly used (31)P-MT method, sequences such as inversion transfer (IT) with short pulses might be better suited for the specific hardware and software limitations of a clinical scanner. In addition, small NMR-undetectable metabolite pools can transfer MT to NMR-visible pools during long saturation pulses, which is prevented with short pulses. (31)P-MT sequences were adapted for limited pulse length, for heteronuclear transmit-receive surface coils with inhomogeneous B1 , for the need for volume selection and for the inherently low signal-to-noise ratio (SNR) on a clinical 3-T MR system. The ST and IT sequences were applied to skeletal muscle and liver in 10 healthy volunteers. Monte-Carlo simulations were used to evaluate the behavior of the IT measurements with increasing imperfections. In skeletal muscle of the thigh, ATP synthesis resulted in forward reaction constants (k) of 0.074 ± 0.022 s(-1) (ST) and 0.137 ± 0.042 s(-1) (IT), whereas the creatine kinase reaction yielded 0.459 ± 0.089 s(-1) (IT). In the liver, ATP synthesis resulted in k = 0.267 ± 0.106 s(-1) (ST), whereas the IT experiment yielded no consistent results. ST results were close to literature values; however, the IT results were either much larger than the corresponding ST values and/or were widely scattered. To summarize, ST and IT experiments can both be implemented on a clinical body scanner with heteronuclear transmit-receive surface coils; however, ST results are much more robust against experimental imperfections than the current implementation of IT.

Using the Morse Potential to Understand Reaction Coördinates using Maple

The interactions employed in the “linear” reaction A(g)+BC(g) -> AB(g) + C(g) in a “one dimensional world” can be used to illustrate the “reaction coördinate”, using Maple, in a manner which allows students to inspect potential energy surfaces, make contour maps of those surfaces, and conceptually construct the “reaction coördinate” by tracing the local minimum path on the surface created.“one dimensional world” can be used to illustrate the “reaction coördinate”, using Maple, in a manner which allows students to inspect potential energy surfaces, make contour maps of those surfaces, and conceptually construct the “reaction coördinate” by tracing the local minimum path on the surface created.

Heterocyst glycolipids in surface sediments and water samples of the Amazon shelf

Marine endosymbiotic heterocystous cyanobacteria make unique heterocyst glycolipids (HGs) containing pentose (C5) moieties. Functionally similar HGs with hexose (C6) moieties found in free-living cyanobacteria occur in the sedimentary record, but C5 HGs have not been documented in the natural environment. Here we developed a high performance liquid chromatography multiple reaction monitoring (MRM) mass spectrometry (HPLC-MS2) method specific for trace analysis of long chain C5HGs and applied it to cultures of Rhizosolenia clevei Ostenfeld and its symbiont Richelia intracellularis which were found to contain C5 HGs and no C6 HGs. The method was then applied to suspended particulate matter (SPM) and surface sediment from the Amazon plume region known to harbor marine diatoms carrying heterocystous cyanobacteria as endosymbionts. C5 HGs were detected in both marine SPM and surface sediments, but not in SPM or surface sediment from freshwater settings in the Amazon basin. Rather, the latter contained C6 HGs, established biomarkers for free-living heterocystous cyanobacteria. Our results indicate that the C5 HGs may be potential biomarkers for marine endosymbiotic heterocystous cyanobacteria.

Pigment concentrations in surface sediments of the Arabian Sea

As part of the large-scale, interdisciplinary deep-sea study "BIGSET", the relationship between the monsoon-induced regional and temporal variability of POC deposition and the small-sized benthic community was investigated at several sites 2316-4420 m deep in the Arabian Sea during four cruises between 1995 and 1998. Vertical and horizontal distribution patterns of chloroplastic pigments (a measure of phytodetritus deposition), readily soluble protein and activity, and biomass parameters of the small-sized benthic community (Electron Transport System Activity (ETSA); bacterial ectoenzymatic activity (FDA turnover) and DNA concentrations) were measured concurrently with the vertical fluxes of POC and chloroplastic pigments. Sediment chlorophyll a (chl. a) profiles were used to calculate chl. a flux rates and to estimate POC flux across the sediment water interface using two different transport reaction models. These estimates were compared with corresponding flux rates determined in sediment traps. Regional variability of primary productivity and POC deposition at the deep-sea floor creates a trophic gradient in the Arabian Basin from the NW to the SE, which is primarily related to the activity of monsoon winds and processes associated with the topography of the Arabian Basin and the vicinity of land masses. Inventories of sediment chloroplastic pigments closely corresponded to this trophic gradient. For ETSA, FDA and DNA, however, no clear coupling was found, although stations WAST (western Arabian Sea) and NAST (northern Arabian Sea) were characterised by high concentrations and activities. These parameters exhibited high spatial and temporal variability, making it impossible to recognise clear mechanisms controlling temporal and spatial community patterns of the small-sized benthic biota. Nevertheless, the entire Arabian Basin was recognised as being affected by monsoonal activity. Comparison of two different transport reaction models indicates that labile chl. a buried in deeper sediment layers may escape rapid degradation in Arabian deep-sea sediments.

Porewater and solid-phase phosphorus geochemistry in surface sediments of sediment core GeoB9510-3, GeoB9518-4 and GeoB9519-6

Despite intensive research on the different domains of the marine phosphorus (P) cycle during the last decades, frequently discussed open questions still exist especially on controlling factors for the benthic behaviour of P and its general distribution in sediment-pore water systems. Steady state or the internal balance of all relevant physical and (bio)geochemical processes are amongst the key issues. In this study we present and discuss an extended data set from surface sediments recovered from three locations on the NW African continental slope. Pore water data and results from sequential sediment extractions give clear evidence to the well-known close relationship between the benthic cycles of P and iron. Accordingly, most of the dissolved phosphate must have been released by microbially catalyzed reductive dissolution of iron (oxhydr)oxides. However, rates of release and association of P and iron, respectively, are not directly represented in profiles of element specific sediment compositions. Results from steady-state based transport-reaction modelling suggest that particle mixing due to active bioturbation, or rather a physical net downward transport of P associated to iron (oxyhydr)oxides, is an essential process for the balance of the inspected benthic cycles. This study emphasizes the importance of balancing analytical data for a comprehensive understanding of all processes involved in biogeochemical cycles.

Communication: Transition State Theory for dissipative systems without a dividing surface

Transition state theory is a central cornerstone in reaction dynamics. Its key step is the identification of a dividing surface that is crossed only once by all reactive trajectories. This assumption is often badly violated, especially when the reactive system is coupled to an environment. The calculations made in this way then overestimate the reaction rate and the results depend critically on the choice of the dividing surface. In this Communication, we study the phase space of a stochastically driven system close to an energetic barrier in order to identify the geometric structure unambiguously determining the reactive trajectories, which is then incorporated in a simple rate formula for reactions in condensed phase that is both independent of the dividing surface and exact.

Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif

Surface proteins of Staphylococcus aureus are linked to the bacterial cell wall by sortase, an enzyme that cleaves polypeptides at the threonine of the LPXTG motif. Surface proteins can be released from staphylococci by treatment with hydroxylamine, resulting in the formation of threonine hydroxamate. Staphylococcal extracts, as well as purified sortase, catalyze the hydroxylaminolysis of peptides bearing an LPXTG motif, a reaction that can be inhibited with sulfhydryl-modifying reagents. Replacement of the single conserved cysteine at position 184 of sortase with alanine abolishes enzyme activity. Thus, sortase appears to catalyze surface-protein anchoring by means of a transpeptidation reaction that captures cleaved polypeptides as thioester enzyme intermediates.