Biomedical subjects

Ingo Klimant

Publications and source records attributed to Ingo Klimant.

18 recordsLinked to original sources

PubMed2007

Time-resolved pH/pO2 mapping with luminescent hybrid sensors.

A method for simultaneous and referenced 2D mapping of pH and pO2 is described. The experimental setup combines a fast gateable CCD camera as detector, a LED as excitation light source and a single-layer sensor membrane as optical transducer. The planar optode comprises a lipophilic fluorescein derivative (lifetime approximately 5 ns) and platinum(II) mesotetrakis(pentafluorophenyl)porphyrin (approximately 70 micros in the absence of a quencher) immobilized in a hydrogel matrix. Depending on the fluorescent pH indicator, a pH transition in the physiological range (pH 6-pH 8) or in the near-basic region (pH 7-pH 9) can be achieved. The measuring scheme involves the time-resolved acquisition of images in three windows during a series of square-shaped excitation pulses. A method allowing the calculation of both parameters from these three images is presented. The pH/pO2 hybrid sensor incorporating the pH indicator 2',7'-dihexyl-5(6)-N-octadecyl-carboxamidofluorescein was characterized in detail. The pH and pO2 were determined with a maximum deviation of 0.03 pH unit and 6.5 hPa pO2, respectively, within the range of pH 7.6-pH 8.7 and 0-200 hPa pO2 in test measurements. The ionic strength (IS) cross-sensitivity was found to be relatively small (pH/IS < 3.5 x 10(-4) mM(-1) and pO2/IS <-0.053 hPa mM(-1) at a transition from 0.5 to 0.1 M IS). Whereas a strong temperature effect on the sensor signal was observed (DeltapH/DeltaT = 0.011-0.034 K-1 and DeltapO2/DeltaT = 1.85-7.17 hPa K-1 in the range from 277 to 308 K). Examples of pH/pO2 images obtained in natural marine sediment are presented.

Journal Article↗

PubMed2006

Micro-colony array based high throughput platform for enzyme library screening.

Enzymes are becoming increasingly important tools for synthesizing and modifying fine and bulk chemicals. The availability of biocatalysts which fulfil the requirements of industrial processes is often limited. Recruiting suited enzymes from natural (e.g. metagenomes) and artificial (e.g. directed evolution) biodiversity is based on screening libraries of microbial clones expressing enzyme variants. However, exploring the complex diversity of such libraries needs efficient screening methods. Overcoming the "screening bottleneck" requires rapid high throughput technology allowing the analysis of a large diversity of different enzymes and applying different screening conditions. Facing these facts an efficient and cost effective method for high throughput screening of large enzyme libraries at the colony level was developed. Therefore, ordered high density micro-colony arrays were combined with optical sensor technology and automated image analysis. The system generally allows the simultaneous monitoring of enzyme activities reflected by up to 7000 micro-colonies spotted on a filter in the size of a micro-titer plate. A developed replica option also allows the analysis of clones under varying external conditions. The method was verified by a model screening using esterases and was proved to provide reliable enzyme activity measurements within single micro-colonies allowing the discrimination of activity differences in the range of 10-20%.

Calibration↗

PubMed2006

Fiber-optic flow-through sensor for online monitoring of glucose.

A new microdialysis-based glucose-sensing system with an integrated fiber-optic hybrid sensor is presented. Design and dimensions of the cell are adapted for its coupling with commercially available microdialysis techniques, thereby providing a new system for continuous glucose monitoring. The glucose level is detected via oxygen consumption which occurs as a consequence of enzymatic reaction between immobilized glucose oxidase and glucose. The use of gas-permeable Tygon tubing ensures complete and constant air-saturation of the measuring fluid in the cell. Nevertheless, a reference oxygen optode is used to detect and to compensate response changes caused by events like bacterial growth, temperature fluctuations, or failure of the peristaltic pump. In contrast to widely used electrochemical sensors, the response of the microdialysis-based fiber-optic glucose sensor is highly selective, making this sensor approach particularly advantageous for continuous glucose monitoring of patients in intensive care units. The effects of flow rate, pH, temperature, and common interferences on the sensor response are presented and discussed in detail. The sensor is evaluated in vitro using a 3-day continuous test in glucose-spiked plasma. The ability to measure glucose in humans is demonstrated by coupling the flow-through cell and commercially available microdialysis catheter CMA60. A 24-h monitoring test using this setup is successfully applied to a healthy volunteer.

Enzymes, Immobilized↗

PubMed2006

Modified dual lifetime referencing method for simultaneous optical determination and sensing of two analytes.

Simultaneous fluorometric sensing of two analytes becomes possible using a modified dual lifetime referencing (m-DLR) method. In this scheme, two luminescent indicators are needed that have overlapping absorption and emission spectra but largely different decay times. They are excited by a single light source, and both emissions are measured simultaneously. In the frequency domain m-DLR method, the phase of the short-lived fluorescence of a first indicator is referenced against that of the long-lived luminescence of the second indicator. The analytical information is obtained by measurement of the phase shifts at two modulation frequencies. The method is demonstrated to work for the case of dually sensing oxygen and carbon dioxide. It benefits from simple instrumentation and optical setup. The approach is perceived to be of wide applicability. Examples include (1) analysis of two luminescent analytes, (2) analytical determinations that make use of two probes, and (3) sensing of two species such as carbon dioxide and oxygen (as demonstrated here), or oxygen and chlorophyll, provided the luminophores meet the condition of having largely different decay times and overlapping absorption and emission spectra.

Carbon Dioxide↗

PubMed2005

Gas sensing in microplates with optodes: influence of oxygen exchange between sample, air, and plate material.

Microplates with integrated optical oxygen sensors are a new tool to study metabolic rates and enzyme activities. Precise measurements are possible only if oxygen exchange between the sample and the environment is known. In this study we quantify gas exchange in plastic microplates. Dissolved oxygen was detected using either an oxygen-sensitive film fixed at the bottom of each well or a needle-type sensor. The diffusion of oxygen into wells sealed with different foils, paraffin oil, and paraffin wax, respectively, was quantified. Although foil covers showed the lowest oxygen permeability, they include an inevitable gas phase between sample and sealing and are difficult to manage. The use of oil was found to be critical due to the extensive shaking caused by movement of the plates during measurements in microplate readers. Thus, paraffin wax was the choice material because it avoids convection of the sample and is easy to handle. Furthermore, without shaking, significant gradients in pO2 levels within a single well of a polystyrene microplate covered with paraffin oil were detected with the needle-type sensor. Higher pO2 levels were obtained near the surface of the sample as well as near the wall of the well. A significant diffusion of oxygen through the plastic plate material was found using plates based on polystyrene. Thus, the location of a sensor element within the well has an effect on the measured pO2 level. Using a sensor film fixed on the bottom of a well or using a dissolved pO2-sensitive indicator results in pO2 offset and in apparently lower respiration rates or enzyme activities. Oxygen diffusion through a polystyrene microplate was simulated for measurements without convection--that is, for samples without oxygen diffusion through the cover and for unshaken measurements using permeable sealings. This mathematical model allows for calculation of the correct kinetic parameters.

Computer Simulation↗

PubMed2005

pH fluorosensors for use in marine systems.

In this work we present optical pH sensors especially designed for pH measurements in marine environment. Embedded in an uncharged, highly proton-permeable hydrogel matrix, the two novel lipophilic carboxyfluorescein derivatives 2',7'-dihexyl-5(6)-N-octadecyl-carboxamidofluorescein (DHFA) and 2',7'-dihexyl-5(6)-N-octadecyl-carboxamidofluorescein ethyl ester (DHFAE) have apparent dissociation constants of ca. 8.4. The pH transition range of the sensors perfectly matches the pH range occurring in seawater and marine sediment (ca. pH 7.2-9.2). The cross-sensitivity towards ionic strength (IS) was found to be low for DHFA-containing membranes and was even negligible when using DHFAE as indicator. The quantum yield (QY) of DHFA (0.94(basic)) is similar to that of fluorescein (0.97(basic)). QYs of 0.62(basic) and 0.22(acidic) were found for DHFAE. The optical properties of the indicators enable referenced measuring schemes. Lactonisation of the DHFAE chromophore is prevented by esterification of the carboxyl group in 2 position. Thus, internally referenced dual wavelength measurements are possible since the emission maxima of the basic and acidic form of DHFAE differ by 30 nm. Dual lifetime referenced (DLR) measurements were made with pH sensors incorporating ruthenium-(II)-tris-4,7-diphenyl-1,10-phenanthroline (Ru(dpp)(3))-containing reference particles in addition to the indicator. This type of sensor can be applied for pH imaging or in phase-modulation measurements of pH.

Fluoresceins↗

PubMed2005

In vivo phosphorescence imaging of pO2 using planar oxygen sensors.

OBJECTIVE: Oxygen-dependent quenching of luminescence of metal porphyrin complexes has been used to image the pO(2) distribution over tumor and normal tissue. METHODS: An experimental setup is described using a platinum(II)-octaethyl-porphyrin immobilized in a polystyrene matrix as transparent planar sensor. RESULTS: Sensitivity over a broad range is high at low pO(2) values (+/- 0.2 mm Hg at 0 mm Hg; +/- 1.5 mm Hg at 160 mm Hg pO(2)). Due to intrinsically referencing via lifetime encoding there was no modification of the sensor response in vivo in the dorsal skinfold chamber model with amelanotic melanoma (A-MEL-3) in awake hamsters when compared to the in vitro calibration. pO(2) measurements over normal tissue (25.8 +/- 5.1 mm Hg) and tumor tissue (9.2 +/- 5.1 mm Hg) were in excellent agreement with previous results obtained in this model using a surface multiwire electrode. CONCLUSIONS: Using the presented method the surface pO(2) distribution can be mapped with a high temporal resolution of approximately 100 ms and a spatial resolution of at least 25 mu m. Moreover, the transparent sensor allows the simultaneous visualization of the underlying microvasculature.

Animals↗

PubMed2005

An optical multifrequency phase-modulation method using microbeads for measuring intracellular oxygen concentrations in plants.

A technique has been developed to measure absolute intracellular oxygen concentrations in green plants. Oxygen-sensitive phosphorescent microbeads were injected into the cells and an optical multifrequency phase-modulation technique was used to discriminate the sensor signal from the strong autofluorescence of the plant tissue. The method was established using photosynthesis-competent cells of the giant algae Chara corallina L., and was validated by application to various cell types of other plant species.

Chara↗

PubMed2004

Fluorescent pH sensors with negligible sensitivity to ionic strength.

Optical pH determination has the fundamental disadvantage of measuring a signal that depends on the ionic strength of the sample. The problem originates from the complex relationship between the proton activity and the concentration of the pH-sensitive dye. The effect of ionic strength on the signal depends on the charge of the indicator and its environment, e.g. the immobilisation matrix. We present novel lipophilic fluorescein esters carrying one negative charge. They are embedded in an uncharged, highly proton-permeable hydrogel to give optical pH sensors that show a negligible cross-sensitivity towards ionic strength. The fluorescent dyes differ in their substituents. This variation of substituents results in dissociation constants between 5.5 and 8.5. The indicators were made lipophilic by esterification of the carboxy group with a C(18) alkyl chain. Since their spectral properties are quite similar, two indicators may be used in one sensor. This results in an optical pH sensor with a dynamic range that extends from pH 4.5 to 8.

Journal Article↗

PubMed2003

Cross-reactive metal ion sensor array in a micro titer plate format.

A cross-reactive array in a micro titer plate (MTP) format is described that is based on a versatile and highly flexible scheme. It makes use of rather unspecific metal ions probes having almost identical fluorescence spectra, thus enabling (a) interrogation at identical analytical wavelengths, and (b) imaging of the probes contained in the wells of the MTP using a CCD camera and an array of blue-light-emitting diodes as a light source. The unselective response of the indicators in the presence of mixtures of five divalent cations generates a characteristic pattern that was analyzed by chemometric tools. The fluorescence intensity of the indicators was transferred into a time-dependent parameter applying a scheme called dual lifetime referencing. In this method, the fluorescence decay profile of the indicator is referenced against the phosphorescence of an inert reference dye added to the system. The intrinsically referenced measurements also were performed using blue LEDs as light sources and a CCD camera without intensifiers as the detector. The best performance was observed if each well was excited by a single LED. The assembly allows the detection of dye concentrations in the nanomoles-per-liter range without amplification and the acquisition of 96 wells simultaneously. The pictures obtained form the basis for evaluation by pattern recognition algorithms. Support vector machines are capable of predicting the presence of significant concentrations of metal ions with high accuracy.

Journal Article↗

PubMed2003

Integrated optical sensing of dissolved oxygen in microtiter plates: a novel tool for microbial cultivation.

Microtiter plates with integrated optical sensing of dissolved oxygen were developed by immobilization of two fluorophores at the bottom of 96-well polystyrene microtiter plates. The oxygen-sensitive fluorophore responded to dissolved oxygen concentration, whereas the oxygen-insensitive one served as an internal reference. The sensor measured dissolved oxygen accurately in optically well-defined media. Oxygen transfer coefficients, k(L)a, were determined by a dynamic method in a commercial microtiter plate reader with an integrated shaker. For this purpose, the dissolved oxygen was initially depleted by the addition of sodium dithionite and, by oxygen transfer from air, it increased again after complete oxidation of dithionite. k(L)a values in one commercial reader were about 10 to 40 h(-1). k(L)a values were inversely proportional to the filling volume and increased with increasing shaking intensity. Dissolved oxygen was monitored during cultivation of Corynebacterium glutamicum in another reader that allowed much higher shaking intensity. Growth rates determined from optical density measurement were identical to those observed in shaking flasks and in a stirred fermentor. Oxygen uptake rates measured in the stirred fermentor and dissolved oxygen concentrations measured during cultivation in the microtiter plate were used to estimate k(L)a values in a 96-well microtiter plate. The resulting values were about 130 h(-1), which is in the lower range of typical stirred fermentors. The resulting maximum oxygen transfer rate was 26 mM h(-1). Simulations showed that the errors caused by the intermittent measurement method were insignificant under the prevailing conditions.

Bioreactors↗

PubMed2003

PH-sensing 96-well microtitre plates for the characterization of acid production by dairy starter cultures.

A new method for characterization of acid production by dairy starter cultures is presented. Microplates with integrated optical pH sensors are developed. Two fluorophores, a pH-sensitive and a pH-insensitive one are immobilised at the bottom of a polystyrene 96-well microtitre plate. The pH-insensitive fluorophore serves as an internal reference and makes calibration unnecessary. The sensor measures pH accurately in optically well-defined media. Particles and fluorophores contained in the bulk medium disturbed the measurements. Despite these disturbances it was possible to clearly sense differences in inoculum type and in inoculum sizes of cultures of Lactococcus lactis and of Streptococcus thermophilus at 30 and 37 degrees C. Besides a pH-related signal there is information about other changes during milk fermentation. The cultivation results were compared with those from the established CINAC-method. From this comparison it can be concluded that the new method can be used reliably to characterize particularly a large number of strains for screening purposes but also for quality control.

Animals↗

PubMed2002

Determination of oxygen gradients in engineered tissue using a fluorescent sensor.

Nutrient and oxygen supply of cells are crucial to tissue engineering in general. If a sufficient supply cannot be maintained, the development of the tissue will slow down or even fail completely. Previous studies on oxygen supply have focused on measurement of oxygen partial pressures (pO(2)) in culture media or described the use of invasive techniques with spatially limited resolution. The experimental setup described here allows for continuous, noninvasive, high-resolution pO(2) measurements over the cross-section of cultivated tissues. Applying a recently developed technique for time-resolved pO(2) sensing using optical sensor foils, containing luminescent O(2)-sensitive indicator dyes, we were able to monitor and analyze gradients in the oxygen supply in a tissue over a 3-week culture period. Cylindrical tissue samples were immobilized on top of the sensors. By measuring the luminescence decay time, two-dimensional pO(2) distributions across the tissue section in contact with the foil surface were determined. We applied this technique to cartilage explants and to tissue-engineered cartilage. For both tissue types, changes were detected in monotonously decreasing gradients of pO(2) from the surface with high pO(2) to minimum pO(2) values in the center of the samples. Nearly anoxic conditions were observed in tissue constructs ( approximately 0 Torr) but not in excised cartilage discs ( approximately 20 Torr) after 1 day. Furthermore, the oxygen supply seemed to strongly depend on cell density and cell function. Additionally, histological analysis revealed a maximum depth of approximately 1.3 mm of regular cartilage development in constructs grown under the applied culture conditions. Correlating analytical and histological analysis with the oxygen distributions, we found that pO(2) values below 11 Torr might impair proper tissue development in the center. The results illustrate that the method developed is an ideal one to precisely assess the oxygen demand of cartilage cultures.

Animals↗

PubMed2002

Fiber optic multi-channel protein detector for use in preparative continuous annular chromatography.

Continuous annular chromatography is an effective method in the separation of preparative scale quantities of biological compounds including proteins where established batch chromatography borders on it. The need for identification or quantification of proteins triggered the development of respective detection units. Here, we describe two types of optical multi-channel detectors. The first is a fiber optic multi-channel detector suitable for the separation of aqueous protein solutions. The second is a technically improved (circular optic) device suitable for application in multi-channel detection. Specifically, UV-absorption measurements of proteins at 280 nm were carried out using newly designed fiber optic detectors having eight and 16 channels. Calibration plots were established for a series of stock solutions of known concentrations of proteins. Mathematical functions were derived from these calibration data to simulate the response of the detector. Limits of detection and the ranges of validity of the fit functions were determined. The 16-channel detector has a theoretical limit of detection that is equivalent to absorbance changes of 10(-4) units.

Chromatography, Liquid↗

PubMed2002

Homogeneous luminescence decay time-based assay using energy transfer from nanospheres.

Following a study on the feasibility of resonance energy transfer (RET) from carboxylated nanospheres with an incorporated phosphorescent donor to a cationic polyelectrolyte/acceptor aggregate on their surface, a novel scheme for homogeneous assays is presented that is based on RET from phosphorescent biotinylated nanospheres to fluorescently labeled streptavidin (SA). The phosphorescent nanospheres, with a diameter of well below 50 nm, are made from carboxylated polyacrylonitrile and dyed with ruthenium(II)-tris-4,7-diphenyl-1,10-phenanthroline dichloride (Ru(dpp)). Due to the small size of the nanospheres and the complete extraction of the ruthenium dye into the nanospheres during the precipitation process, RET occurs from Ru(dpp) to the label if labeled SA binds to the surface of the nanospheres. Luminescence quenching by oxygen or other species present in the sample can be neglected due to the shielding effect of the polymer matrix. Based on this finding, a competitive binding assay was established, where avidin and labeled SA compete for the biotin binding sites on the nanosphere. The process of binding to the surface can be detected by measurement of the luminescence intensity or the apparent decay time which is in the order of 2.5-4.5 micros.

Biotinylation↗

PubMed2002

Sol-gel based optical carbon dioxide sensor employing dual luminophore referencing for application in food packaging technology.

An optical sensor for the measurement of carbon dioxide in Modified Atmosphere Packaging (MAP) applications has been developed. It is based on the fluorescent pH indicator 1-hydroxypyrene-3,6,8-trisulfonate (HPTS) immobilised in a hydrophobic organically modified silica (ormosil) matrix. Cetyltrimethylammonium hydroxide was used as an internal buffer system. Fluorescence is measured in the phase domain by means of the Dual Luminophore Referencing (DLR) sensing scheme which provides many of the advantages of lifetime-based fluorometric sensors and makes it compatible with established optical oxygen sensor technology. The long-term stability of the sensor membranes has been investigated. The sensor displays 13.5 degrees phase shift between 0 and 100% CO2 with a resolution of better than 1% and a limit of detection of 0.08%. Oxygen cross-sensitivity is minimised (0.6% quenching in air) by immobilising the reference luminophore in polymer nano-beads. Cross-sensitivity towards chloride and pH was found to be negligible. Temperature effects were studied, and a linear Arrhenius correlation between ln k and 1/T was found. The sensor is stable over a period of at least seven months and its output is in excellent agreement with a standard reference method for carbon dioxide analysis.

Carbon Dioxide↗

PubMedDate not supplied

Modeling of mixing in 96-well microplates observed with fluorescence indicators.

Mixing in 96-well microplates was studied using soluble pH indicators and a fluorescence pH sensor. Small amounts of alkali were added with the aid of a multichannel pipet, a piston pump, and a piezoelectric actuator. Mixing patterns were observed visually using a video camera. Addition of drops each of about 1 nL with the piezoelectric actuator resulted in umbrella and double-disklike shapes. Convective mixing was mainly observed in the upper part of the well, whereas the lower part was only mixed quickly when using the multichannel pipet and the piston pump with an addition volume of 5 microL or larger. Estimated mixing times were between a few seconds and several minutes. Mixing by liquid dispensing was much more effective than by shaking. A mixing model consisting of 21 elements could describe mixing dynamics observed by the dissolved fluorescence dye and by the optical immobilized pH sensor. This model can be applied for designing pH control in microplates or for design of kinetic experiments with liquid addition.

Acids↗

PubMedDate not supplied

A combinatorial approach for development of materials for optical sensing of gases.

We present a combinatorial approach for development of materials for use in optical gas sensors, with oxygen being used as an exemplary target gas. Combinatorial chemistry is shown to be a promising tool for speeding up the search for new sensor materials. The method is based on the use of various polymers, solvents, indicators, plasticizers, and other additives. Solutions of the respective materials are prepared in appropriate organic solvents, and a robotic station is programmed to mix the components. Spots of the sensing materials are deposited in the wells of glass substrates resembling microtiterplates. After drying off the solvent, the sensor spots are automatically analyzed in a test stand, where they are exposed to a carrier gas containing oxygen in various concentrations. Changes in the decay time of fluorescence of the indicator probes are measured and used (along with sensor response time) as a main criterion for sensor assessment. It is shown that the combinatorial approach can reduce the time and effort needed to establish libraries of sensor materials by a factor of at least 1000. We describe in detail the device for preparation of sensor libraries and for testing the respective materials. The potential of the system is demonstrated for the characterization of optical oxygen sensors.

Journal Article↗