New optical biosensors for uric acid and glucose

This thesis describes the development of a microtiter plate assay for determination of uric acid. It also depicts the development, characterization and application of luminescence based optical biosensors for determination of uric acid and glucose. Chapter 1 gives an introduction on the importance of the determination of uric acid and glucose. Furthermore, an overview on the state of the art of optical sensing of oxygen and pH is given, along with a comparison of optical sensor versus electrochemical sensor technology. In this thesis a microtiter plate assay for uric acid determination is described applying an europium complex as fluorescent probe. Hence, the luminescence emission mechanism of lanthanide complexes is explained in detail. Chapter 2 describes a microtiter plate assay for uric acid. The hydrogen peroxide sensitive probe europium(III)-tetracycline (Eu3TC) was applied for sensing hydrogen peroxide that is released by the enzyme uricase during the oxidation of uric acid. Hydrogen peroxide coordinates to Eu3Tc and enhances its luminescence intensity. The assay is carried out in the time-gated mode. Uric acid can be detected in the concentration range up to 60 µM with a limit of detection of 9.9 µM. The assay cannot be applied to the determination of uric acid in urine unfortunately. Urine a complex matrix contains phosphate which quenches the luminescence intensity of the complex Eu3TC-HP strongly. In chapter 3, a biosensor membrane is presented for detection of uric acid. The biosensor membrane consists of a single layer which contains an oxygen probe and the enzyme uricase. The detection of uric acid is based on the measurement of oxygen that is consumed during the oxidation catalyzed by uricase. A ruthenium- or an iridium complex incorporated in organically modified sol-gel is applied as oxygen sensitive probe, whose luminescence intensity is dynamically quenched in presence of oxygen. Uric acid can be detected in the concentration range up to 0.8 mM with a limit of detection of 0.05 mM using the ruthenium complex as oxygen sensitive probe. Applying the iridium complex as oxygen sensitive probe uric acid is detected in the concentration range from 0.02 to 0.6 mM. The optical biosensor using the ruthenium complex as oxygen sensitive probe is successfully applied to the determination of uric acid in blood serum. In chapter 4, a biosensor membrane is prepared for determination of glucose. The fundamental idea of this chapter is the simultaneous detection of glucose via (a) oxygen and (b) pH transduction. The first part of this chapter describes a strategy to embed an oxygen sensitive probe and the enzyme glucose oxidase in a hydrogel matrix and apply it to glucose sensing. The detection scheme is based on the principle described in chapter 3. The enzyme glucose oxidase catalyzes the oxidation of glucose under oxygen consumption which is detected using a ruthenium complex as the oxygen sensitive probe. Its luminescence intensity is enhanced in absence of oxygen. Glucose can be detected in the concentration range from 0.2 to 1.0 mM. In the second part glucose is determined via a pH transducer. During the oxidation of glucose one of the end products is gluconic acid which dissociates in gluconate and protons. Hence, the pH of the microenvironment decreases which can be detected in luminescence changes of a pH sensitive probe. Two pH indicators, HPTS and CF, are applied but without the desirable success. The application of CF as pH indicator is disadvantageous due to strong photobleaching. However, the determination of glucose using HPTS as pH transducer is feasible only in the molar concentration range and the reproducibility of the results is very low. Hence, the simultaneous determination of glucose via oxygen and pH transduction is not possible. In chapter 5, a triple sensor is presented for simultaneous monitoring of glucose via an oxygen and pH transducer along with monitoring the temperature. The oxygen-, pH- and temperature transducers are embedded in a hydrogel matrix along with the enzyme glucose oxidase. The triple sensor is illuminated by one single LED and the resulting emissions of the indicators are imaged by a CCD camera and spectrally separated by using suitable filters. This set up allows the simultaneous monitoring of glucose via oxygen and pH transduction and the temperature. This temperature determination is important because the activity of the enzyme GOx and the luminescence of the oxygen transducer PtTFPL are temperature dependent. Simultaneous sensing of glucose via pH and oxygen transduction along with temperature is successful in the case of monitoring glucose via oxygen transduction along with the temperature. The detection of glucose via pH transduction lacks the desirable success because the response times are very long and the reproducibility of the results is very low

Fully reversible optical biosensors for uric acid using oxygen transduction

An optical biosensor is presented for continuous determination of uric acid. The scheme is based on the measurement of the consumption of oxygen during the oxidation of uric acid that is catalyzed by the enzyme uricase. The enzyme is immobilized in a polyurethane hydrogel next to a metal-organic probe whose fluorescence is quenched by oxygen. The consumption of oxygen was followed by measurement of changes of luminescence intensity of two kind of probes and can be related to the concentration of uric acid. Analytical ranges (0–2 mM), the response times (80–100 s), reproducibility, and long-term stability were investigated. The biosensors are stable for at least 1 month and are not interfered by common interferents. One kind of biosensor was applied to the determination of uric acid in human blood serum. The results agree with those of a commercial colorimetric detection kit

Time-Resolved Fluorescence-Based Assay for the Determination of Alkaline Phosphatase Activity and Application to the Screening of Its Inhibitors

A single-step end point method is presented for determination of the activity of the enzyme alkaline phosphatase (ALP) using the effect of enhancement of fluorescence of the easily accessible europium(III)-tetracycline 3:1 complex (Eu3TC). Its luminescence, peaking at 616 nm if excited at 405 nm, is enhanced by a factor of 2.5 in the presence of phosphate. Phenyl phosphate was used as a substrate that is enzymatically hydrolyzed to form phenol and phosphate. The latter coordinates to Eu3TC and enhances its luminescence intensity as a result of the displacement of water from the inner coordination sphere of the central metal. The assay is performed in a time-resolved (gated) mode, which is shown to yield larger signal changes than steady-state measurement of fluorescence. The limit of detection for ALP is 4 µmol L—1. Based on this scheme, a model assay for theophylline as inhibitor for ALP was developed with a linear range from 14 to 68 µmol L— 1 of theophylline