Optical enzyme-based sensors

Optical Enzyme-Based Sensors for Reagentless Detection of Chemical Analytes, Chapter 4... 

Another approach, developed in our laboratory, consists of the compartmentalization of the sensing layer25"27. This concept, only applicable for multi-enzyme based sensors, consist in immobilizing the luminescence enzymes and the auxiliary enzymes on different membranes and then in stacking these membranes at the sensing tip of the optical fibre sensor. This configuration results in an enhancement of the sensor response, compared with the case where all the enzymes are co-immobilized on the same membrane. This was due to an hyperconcentration of the common intermediate, i.e. the final product of the auxiliary enzymatic system, which is also the substrate of the luminescence reaction, in the microcompartment existing between the two stacked membranes. 

Fuh M.R.S., Burgess L.W., Christian G.D., Single fiber-optic fluorescence enzyme-based sensor, Anal. Chem. 1988 60 433-435. 

Moreno-Bondi MC, Benito-Pena E (2005) Fundamentals of enzyme-based sensors. In Martellucci S, Baldini F (eds) Optical chemical sensors. Springer-Kluwer, New York (in press)... 

Arnold M.A., Enzyme-based fiber optic sensor, Anal. Chem. 1985 57 (2) 565. 

The detection modes commonly applied in enzyme-based optical fiber sensors are based on one of the following principles ... 

Optical transduction modes applied in combination with enzyme based fiber-optic sensors include absorbance, reflectance, fluorescence,... 

Enzyme-based optical sensor applications will be further described in this book. They are still the most widespread optical biosensors but work is needed to overcome limitations such as shelf life, long term stability, in situ measurements, miniaturization, and the marketing of competitive devices. 

Figure 8. Examples of enzyme-based fiber-optic sensor designs. Adapted from refs, a) 43 b) 44 c) 45 d) 46 e) 47 f) 48.

Freeman and Seitz [6] developed one of the first enzyme-based CL sensors with convincing performance. They immobilized horseradish peroxidase (HRP) at the end of an optical fiber and achieved a detection limit of 2 X 10 4 6 mol/L H202. Preuschoff et al. [23] developed a fiberoptic flow cell for H202 detection with long-term stability, suitable for fast FTA. Different peroxidases were covalently... 

Fig. 9.32 General scheme of enzyme-based optical sensor...

Aizawa presented an overview on the principles and applications of the electrochemical and optical biosensors [61]. The current development in the biocatalytic and bioaffinity bensensor and the applications of these sensors were given. The optical enzyme sensor for acetylcholine was based on use of an optical pH fiber with thin polyaniline film. 

Enzyme sensors can measure analytes that are the substrates of enzymatic reactions. Thermometric sensors can measure the heat produced by the enzyme reaction [31], while optical or electrochemical transducers measure a product produced or cofactor consumed in the reaction. For example, several urea sensors are based on the hydrolysis of urea by urease producing ammonia, which can be detected by an ammonium ion-selective ISE or ISFET [48] or a conductometric device [49]. Amperometric enzyme sensors are based on the measurement of an electroactive product or cofactor [50] an example is the glucose oxidase-based sensor for glucose, the most commercially successful biosensor. Enzymes are incorporated in amperometric sensors in functionalised monolayers [51], entrapped in polymers [52], carbon pastes [53] or zeolites [54]. Other catalytic biological systems such as micro-organisms, abzymes, organelles and tissue slices have also been combined with electrochemical transducers. 

The latter procedure was performed with an electrode for O. An optical sensor was constructed on the immobilised enzyme based on spectrophotometer with optical fibres and probe for oxygen measurement AvaSoft - Oxy, AvaSpec US B1 (Avantes - the Netherlands). 

In a measurement ceU 20 mL of 0.1 Tris-buffer with pH 7.2 the respective sensor was incubated for 3 min with aflatoxin B1 in concentration range of 0.0 5-0.5 (xM previously diluted in dimethyl sulphoxide. After 3 min of incubation at 37°C 0.1 mL hnoleic acid was added to the samples so that the final concentration of the sample was 4 (xM. Based on the measurements, standard curves were constructed for the optical enzyme electrode (Fig. 6). 

---