Norbert Opitz

Optical Fluorescence and Its Application to an Intravascular Blood Gas Monitoring System

Optical fluorescence has an extensive history of application in the laboratory to the measurement of ionic concentrations and the partial pressures of oxygen and carbon dioxide. The use of optical fluorescence based sensors to fulfill a recognized need for continuous invasive monitoring of arterial blood gases offers a number of inherent advantages. However, the requirements placed upon a blood gas probe and supporting instrumentation appropriate for use in the clinical environment result in significant design challenges in selection of suitable fluorescent dyes, maintenance of mechanical integrity while obtaining required miniaturization of sensors, and in the transmission, acquisition, and processing of low level light signals. An optical fluorescence based intravascular blood gas monitoring system has been developed which is particularly suited for the critical care and surgical settings and which has a sensor probe that can be introduced into the patient via a radial artery catheter. This system has shown an excellent agreement of measured with true values of pH, pCO2, and P02 in both in vitro and animal studies. Linear regression analysis of typical in vitro data, where true levels were established via tonometry and standardization to a high accuracy laboratory pH measuring instrument, shows slope/intercept values very close to 1.0/0.0 and correlation coefficients of greater than 0.99 for all three parameters.

Membrane-Mediated Induction and Sorting of K-Ras Microdomain Signaling Platforms

The K-Ras4B GTPase is a major oncoprotein whose signaling activity depends on its correct localization to negatively charged subcellular membranes and nanoclustering in membrane microdomains. Selective localization and clustering are mediated by the polybasic farnesylated C-terminus of K-Ras4B, but the mechanisms and molecular determinants involved are largely unknown. In a combined chemical biological and biophysical approach we investigated the partitioning of semisynthetic fully functional lipidated K-Ras4B proteins into heterogeneous anionic model membranes and membranes composed of viral lipid extracts. Independent of GDP/GTP-loading, K-Ras4B is preferentially localized in liquid-disordered (l(d)) lipid domains and forms new protein-containing fluid domains that are recruiting multivalent acidic lipids by an effective, electrostatic lipid sorting mechanism. In addition, GDP-GTP exchange and, thereby, Ras activation results in a higher concentration of activated K-Ras4B in the nanoscale signaling platforms. Conversely, palmitoylated and farnesylated N-Ras proteins partition into the l(d) phase and concentrate at the l(d)/l(o) phase boundary of heterogeneous membranes. Next to the lipid anchor system, the results reveal an involvement of the G-domain in the membrane interaction process by determining minor but yet significant structural reorientations of the GDP/GTP-K-Ras4B proteins at lipid interfaces. A molecular mechanism for isoform-specific Ras signaling from separate membrane microdomains is postulated from the results of this study.

Interaction of hIAPP with Model Raft Membranes and Pancreatic β‐Cells: Cytotoxicity of hIAPP Oligomers

Type II diabetes mellitus (T2DM) is associated with β‐cell failure, which correlates with the formation of pancreatic islet amyloid deposits. The human islet amyloid polypeptide (hIAPP) is the major component of islet amyloid and undergoes structural changes followed by self‐association and pathological tissue deposition during aggregation in T2DM. There is clear evidence that the aggregation process is accelerated in the presence of particular lipid membranes. Whereas hIAPP aggregation has been extensively studied in homogeneous model membrane systems, especially negatively charged lipid bilayers, information on the interaction of hIAPP with heterogeneous model raft membranes has been missing until now. In the present study, we focus on the principles of aggregation and amyloid formation of hIAPP in the presence of model raft membranes. Time‐lapse tapping mode AFM and confocal fluorescence microscopy experiments followed membrane permeabilization and localization of hIAPP in the raft membrane. Together with the ThT and WST‐1 assay, the data revealed elevated cytotoxicity of hIAPP oligomers on INS‐1E cells.

Fluorescence microscopy studies on islet amyloid polypeptide fibrillation at heterogeneous and cellular membrane interfaces and its inhibition by resveratrol

Type II diabetes mellitus (T2DM) is a disease characterized by progressive deposition of amyloid in the extracellular matrix of β‐cells. We investigated the interaction of the islet amyloid polypeptide (IAPP) with lipid model raft mixtures and INS‐1E cells using fluorescence microscopy techniques. Following preferential partitioning of IAPP into the fluid lipid phase, the membrane suffers irreversible damage and predominantly circularly‐shaped lipid‐containing IAPP amyloid is formed. Interaction studies with the pancreatic β‐cell line INS‐1E revealed that growing IAPP fibrils also incorporate substantial amounts of cellular membranes in vivo. Additionally, the inhibitory effect of the red wine compound resveratrol on IAPP fibril formation has been studied, alluding to its potential use in developing therapeutic strategies against T2DM.

Influence of Enzyme Concentration and Thickness of the Enzyme Layer on the Calibration Curve of the Continuously Measuring Glucose Optode

Since the first construction of enzyme electrodes by Updike and Hicks (1967) and Clark and Sachs (1968) bioelectrodes have been used in physiology, for example for the determination of glucose in blood and serum. One of the main advantages is that measurements can be made continuously for longer times. In these applications an enzyme reaction is coupled to the electrode process. A glucose electrode consists of a membrane of immobilized glucose oxidase, E. C. 1.1.3.4.. The enzyme catalyzes the oxidation of glucose to gluconic acid by dissolved oxygen from blood or serum. The difference in Po2 generated by the enzyme layer is a measure for glucose concentrations. The Clark electrode uses the reaction product H 2O2.

Temperature-pressure phase diagram of a heterogeneous anionic model biomembrane system: Results from a combined calorimetry, spectroscopy and microscopy study

By using Fourier transform infrared (FT-IR) spectroscopy in combination with differential scanning calorimetry (DSC) coupled with pressure perturbation calorimetry (PPC), ultrasound velocimetry, Laurdan fluorescence spectroscopy, fluorescence microscopy and atomic force microscopy (AFM), the temperature and pressure dependent phase behavior of the five-component anionic model raft lipid mixture DOPC/DOPG/DPPC/DPPG/cholesterol (20:5:45:5:25 mol%) was investigated. A temperature range from 5 to 65 °C and a pressure range up to 16 kbar were covered to establish the temperature-pressure phase diagram of this heterogeneous model biomembrane system. Incorporation of 10-20 mol% PG still leads to liquid-ordered (l(o))-liquid-disordered (l(d)) phase coexistence regions over a wide range of temperatures and pressures. Compared to the corresponding neutral model raft mixture (DOPC/DPPC/Chol 25:50:25 mol%), the p,T-phase diagram is - as expected and in accordance with the Gibbs phase rule - more complex, the phase sequence as a function of temperature and pressure is largely similar, however. This anionic heterogeneous model membrane system will serve as a more realistic model biomembrane system to study protein interactions with anionic lipid bilayers displaying liquid-disordered/liquid-ordered domain coexistence over a wide range of the temperature-pressure plane, thus allowing also studies of biologically relevant systems encountered under extreme environmental conditions.

Evidence for redistribution-associated intracellular pK shifts of the pH-sensitive fluoroprobe carboxy-SNARF-1

Properties and peculiarities of the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1 (carboxy-SNARF-1), in view of pHi measurements in single cells, were evaluated using confocal laser scanning microscopy. It was found that in human malignant glioma cells (U 118 MG) grown in multicellular spheroid culture, intracellular calibration curves (nigericin method) varied from one cell to another despite emission ratioing of the fluorescence signals. In addition, considerable deviations between indicator calibration in cell-free solution and intracellular calibration were observed. Microspectrofluorometric measurements revealed that these deviations are attributable to intracellular pK shifts of the indicator rather than to spectral changes of the fluorescence emission. The observed pK shifts are probably due to intracellular redistribution of the indicator between cytosol and lipophilic cell compartemants, e. g. plasma membrane, since the indicator can even be loaded efficiently into the cells via its active acid form (instead of the acetoxymethyl ester form). An approximate theoretical derivation of a cellular calibration curve confirms that a reversible, pH-dependent intracellular redistribution of the protonated indicator component results in an apparent pK shift of ΔpK = log(1 + ɛ¸P with P the partition coefficient and ε a factor that depends on the different mean layer thicknesses of the cytosol and plasma membrane. Since the apparent pK shift amounts to about 1 pH unit in tumour cells of spheroids, the intracellular pH measuring range of carboxy-SNARF-1 is almost restricted to alkaline pH values. Further consequences of the redistribution phenomenon are discussed with special respect to intracellular ion imaging.

Single molecule FCS-based oxygen sensor (O2-FCSensor): a new intrinsically calibrated oxygen sensor utilizing fluorescence correlation spectroscopy (FCS) with single fluorescent molecule detection sensitivity

Abstract We report about a new intrinsically calibrated sensor technology exemplified by means of an O 2 -FCSensor. The O 2 -FCSensor is particularly suited for the quantitative determination of molecular oxygen within biological gases and fluids. The technology is based on the determination of the highly O 2 -dependent triplet transition rates of suitable fluorophores such as Rhodamine Green or Fluorescein. The method utilizes fluorescence correlation spectroscopy (FCS) with the ultimate detection sensitivity of a single fluorescent molecule. Calibration curves of the triplet fraction ( T ) and triplet relaxation time ( τ T ) as a dependence of the oxygen concentration have been determined using a thin (μm) sensor layer, and can be well approximated by hyperbolic sensor characteristics with oxygen sensitivities in the order of 0.1/(%O 2 ). Interestingly, the oxygen sensitivity of triplet fractions of O 2 -FCSensors are not influenced by changes in viscosity of the solution demonstrating that FCS-based O 2 measurements are not diffusion controlled (in contrast to the dynamic process of collisional fluorescence quenching by molecular oxygen). Due to the ultimate sensitivity of this technique it can be applied to nanomolar or even single molecule concentrations of fluorophores thus toxic or cancerogenic influences of the applied indicators are avoided or at least minimized. Furthermore, new fluorescent indicator classes with exceptional photophysical properties can be explored for FCS-based chemo- and biosensors enabling the measurement of other analytes such as, e.g. hydrogen ion activities and carbon dioxide. As a consequence, blood gas analysis (pH, pCO 2 and pO 2 ) may be realized on the basis of intrinsically calibrated FCS-Sensor technology comprising the potential of absolute measurement with unprecedented sensitivity.

Fabrication of 2D-protein arrays using biotinylated thiols: results from fluorescence microscopy and atomic force microscopy

The coupling of a fluorescently labelled protein, streptavidin, to biotinylated organic surfaces is investigated using fluorescence microscopy (FM) and atomic force microscopy (AFM). In order to study the importance of non-specific adsorption investigations were carried out using two-dimensionally structured self-assembled monolayers (SAMs), which were fabricated using micro-contact printing. The relative amount of specific vs. non-specific adsorption could be readily determined by comparing the amount of streptavidin adsorbed on adjacent regions consisting of biotinylated organothiolates and of protein-resistant oligoethyleneglycol (OEG)-thiols.

Quantitative Fluorescence Photometry with Biological Fluids and Gases

Longmuir and Knopp (1) have shown that the quenching of the fluorescence of pyrene butyric acid by oxygen (2) can be used to monitor oxygen concentration in tissue. However, since the fluorescence signal is also influenced by several other factors, such as 1) changes in the indicator concentration, 2) interaction of this indicator with other substances and 3) the filter effect of the tissue, a quantitative evaluation meets with considerable difficulties. The error caused by concentration changes can be cancelled by using special indicators which change their spectra with the reaction. For example, Boldt and Lubbers (3) used s-methyl umbelliferon as a pH indicator which has different excitation spectra for the dissociated and undissociated forms. The other errors cannot be taken into account without special measurements. The actual, possibly disturbed spectrum of the tissue can be related to the true indicator spectrum, if the indicator in the tissue is changed into its dissociated or undissociated form, e. g. on a deliberate change of the pH.