Wolfgang Trettnak

Effects of polymer matrices on calibration functions of luminescent oxygen sensors based on porphyrin ketone complexes.

The design of luminescent oxygen sensors is guided by optimizing sensitivity and/or the form of the calibration function. Both qualities are governed by the molecular processes of luminescence quenching. To evaluate the influence of matrix effects, we prepared membranes based on oxygen-sensitive phosphorescent complexes of porphyrin ketones dissolved in plasticizer-free poly(vinyl chloride) (PVC) and polystyrene (PS). In a PVC matrix, both platinum(II) and palladium(II) octaethylporphyrin ketones exhibited perfectly linear Stern-Volmer intensity plots and almost single-exponential excited state decays. In a PS matrix, the sensitivity of palladium(II) octaethylporphyrin ketone was among the highest reported to date. Yet, slightly nonlinear Stern-Volmer plots and nonexponential decays illustrate the significance of matrix effects of PS. Addition of plasticizers to PVC-based sensors allowed tuning of the oxygen sensitivity in a wide range, while the Stern-Volmer plots became pronouncedly nonlinear. For the plasticizer bis(2-ethylhexyl) adipate, the decay profile was single-exponential in the absence but nonexponential in the presence of oxygen, which is attributed to a distribution of quenching rate constants.

Fully reversible fibre-optic glucose biosensor based on the intrinsic fluorescence of glucose oxidase

Abstract In a new type of glucose biosensor, the intrinsic green fluorescence of glucose oxidase (GOD) is used to provide the analytical information. It was found that the fluorescence of GOD changes during interaction with glucose. Fluorescence is excited at 450 nm and measured at ⪢ 500 nm, which is a wavelength range that is compatible with glass and plastic fibres. The signal response is fully reversible because oxygen is a second substrate. A major feature of this sensor relies on the fact that the recognition element is identical with the transducer element. Enzyme solutions are entrapped at the fibre end within a semipermeable membrane. The change in fluorescence occurs over a small glucose concentration range (typically 1.5–2 mM), the signal at lower and higher glucose levels being unaffected by changes in glucose concentration. Response times of 2–30 min and regeneration times of 1–10 min are observed. Effects of pH and oxygen concentrations are also investigated. To achieve as extended analytical range (e.g., 2.5–10 mM) and shorter response times, kinetic measurements are suggested.

Optical triple sensor for measuring pH, oxygen and carbon dioxide.

A triple sensor unit consisting of opto-chemical sensors for measurement of pH, oxygen and carbon dioxide in bioreactors is presented. The pH and the CO2 sensor are based on the color change of a pH-sensitive dye immobilized on a polymeric support. The resulting changes in absorption are monitored through optical fibers. The oxygen sensor is based on the quenching of the fluorescence of a metal-organic dye. All three sensors are fully LED compatible. The sensitive membranes consist of plastic films and can be stored and replaced conveniently. The sensors are sterilizable with hydrogen peroxide and ethanol. In addition, the pH sensor is steam sterilizable. Accuracy, resolution and reproducibility fulfill the requirements for use in biotechnological applications. Calibration procedures for each sensor are presented. The working principle and the performance of all three sensors are described, with particular emphasis given to their application in bioreactors.

Optical sensors. Part 34. Fibre optic glucose biosensor with an oxygen optrode as the transducer

A biosensor for the continuous determination of glucose is presented. Glucose oxidase was immobilised covalently on a nylon membrane and the consumption of oxygen was measured by following, via fibre optic bundles, the changes in the fluorescence of an oxygen-sensitive dye whose fluorescence is quenched dynamically by molecular oxygen. The dye is dissolved in a very thin silicone membrane placed beneath the enzyme layer. As a result of the oxidation by the enzyme a certain amount of oxygen is consumed. This amount is indicated by the fluorescent dye. The measurements were performed in flowing air-saturated solutions containing 0.1 M pH 7.0 phosphate buffer. The effects of the amount of immobilised enzyme and the thickness of the indicator layer on response times (t90= 1–6 min), analytical ranges (0.1–20 mM) and relative signal changes (up to 26%) were investigated.

Miniaturized luminescence lifetime-based oxygen sensor instrumentation utilizing a phase modulation technique

Optical oxygen sensors are mainly based on the principle of luminescence quenching. In contrast to already existing intensity-based systems, the measurement of the luminescence lifetime provides certain advantages, such as insensitivity to photobleaching or leaching of the dye or changes in the intensity of excitation light. This facilitates the use of simple optical systems or optical fibers. Phase measurement techniques have shown to be a powerful tool for indirect measurement of luminescence lifetimes. Here, dyes with luminescence decay times in the order of tens or hundreds of microseconds allow the use of simple opto-electronic circuitry and low-cost processing electronics. An optical oxygen sensor module has been developed with the dimensions of only 120 X 60 X 30 mm. The instrument is based on the measurement of the decay time of the luminophore by measuring the phase shift between the square-wave excitation and the detected square-wave of the emission coming from the sensor. The instrument is based on semiconductor devices (light-emitting diodes, photodiodes) and may be used for the detection of oxygen in gaseous or liquid samples. Platinum(II) octaethylporphyrin-ketone, which is incorporated in an oxygen-permeable polymer and which has an unquenched lifetime of approximately 60μs, is used as the oxygen-sensitive dye. The typical measurement range of the device is from 0 to 200 hPa oxygen partial pressure with a resolution of <I hPa over the whole measurement range. Measurement accuracy of <±1 hPa has been obtained for periods of 24 h of continuous measurement. The sensor response times t 90 are typically <1 s for gases and 1-5 min for liquid samples.

Fibre-optic glucose sensor with a pH optrode as the transducer

Abstract An enzyme optrode capable of continuously monitoring glucose concentrations is presented. Glucose oxidase was physically immobilised in a sensing layer at the end of a fibre optic light guide. The enzyme catalyses the oxidation of glucose to give gluconic acid, which, in turn, lowers the pH in the microenvironment of the sensor. The enzymatic reaction can therefore be monitored by following the changes in the fluorescence of a pH-sensitive dye incorporated into the sensing layer. Flow-through measurements were performed in buffer solutions of various strength. The response time for 90% of the total signal change was 8–12 min, and 0.1–2mM glucose was found to be detectable. Saturation was reached at glucose concentrations of 2–3 mM.

Recent progress in optical oxygen sensor instrumentation

Optical methods for the determination of dissolved or gaseous oxygen are mainly based on the principle of fluorescence quenching. Measurement schemes have been reported which employ various oxygen-sensitive dyes and bulky instrumentation. Typically, expensive fluorescence spectrometers or fibre-optic photometers have been used, and the applicability of such instruments is rather limited. A system based on low-cost semiconductor devices (light-emitting diodes (LEDS), photodiodes, low-cost analogue and digital components) and new LED-compatible oxygen-sensitive membranes has been developed at our institute. The instrument is capable of determining dissolved or gaseous oxygen and may be calibrated, for example, by a simple two-point calibration procedure with air-saturated and oxygen-free water. Thermostatization of the flow-through cell results in higher measurement accuracy and in a reduced influence of the ambient temperature on the instrument. The overall performance of the oxygen sensor has been investigated, e.g. measurement stability, effectivity of thermostatization, calibration, oxygen diffusion into the measuring cell and excitation feed-through.

A fiberoptic cholesterol biosensor with an oxygen optrode as the transducer.

A biosensor for the continuous optical determination of cholesterol is presented. Cholesterol oxidase is immobilized covalently on a nylon membrane and the consumption of oxygen is measured by following, via fiberoptic bundles, the changes in fluorescence of an oxygen-sensitive dye whose fluorescence is dynamically quenched by molecular oxygen. The dye is dissolved in a very thin silicone membrane placed beneath the enzyme layer. During interaction of the enzyme with cholesterol, oxygen is consumed, which is indicated by the fluorescent dye. At pH 7.25, the analytical range of the sensor is 0.2 to 3 mM and the time to reach a full steady state in a flowing solution ranges from 7 to 12 min.

Fiber-optic remote detection of pesticides and related inhibitors of the enzyme acetylcholine esterase

Abstract We present a novel method for the remote detection of inhibitors (e.g., pesticides) of the enzyme acetylcholine esterase (AChE) with a fiber-optic photometer based exclusively on the use of solid-state opto-electronic components including light-emitting diodes and photodiodes. The method employs a yellow synthetic enzyme substrate which is hydrolyzed by the enzyme to give a blue product. In the presence of an inhibitor, the rate of formation of this blue product is reduced. The resulting signal change is detected via fiber optics and may serve as an alarm.

Study of the performance of an optochemical sensor for ammonia

An optical sensor for ammonia based on ion pairing has been investigated. A pH-sensitive dye (bromophenol blue) was immobilized as an ion pair with cetyltrimethylammonium in a silicone matrix. The colour of the dye changes reversibly from yellow to blue with increasing concentration of ammonia in the sample. The concentration of ammonia can be determined by measuring the transmittance at a given wavelength. All measurements were performed with a dual-beam, solid state photometer. The measurement range is from 6 × 10−7 to 1 × 10−3 M (0.01 to 17 μg ml−1) in 0.1 M sodium phosphate buffer, pH 8. The 90% and 100% response times at a flow rate of 2.5 ml min−1 are 4 min and 10 min, respectively, for a change from 41.9 to 82.5 μM ammonia, or 12 min and 48 min, respectively, for change from 160 to 0 μM ammonia. A continuous drift in signal baseline and ammonia sensitivity limited the measurement stability. The sensor was useful over a period of a few days. The storage stability is more than 10 months (dry). No interference due to pH was observed in the range from pH 5 to pH 9. Sensor performance is seriously affected by amines and cationic detergents. The sensor could be sterilized with 3% hydrogen peroxide or dry heat (90 °C).