Sensing Layers

Later on, such S-layer-based sensing layers were also used in the development of optical biosensors (optodes), where the electrochemical transduction principle was replaced by an optical one [97] (Fig. 10c). In this approach an oxygen-sensitive fluorescent dye (ruthenium(II) complex) was immobilized on the S-layer in close proximity to the glucose oxidase-sensing layer [97]. The fluorescence of the Ru(II) complex is dynamically quenched by molecular oxygen. Thus, a decrease in the local oxygen pressure as a result of... 

Figure 16. SPR measurement experimental set-up. The zoom on the right is a sketch of the sensing layered structure.

Comparing the two optical transduction techniques (absorption or SPR) used in this work, we can conclude that SPR technique appears to be more suitable for gas sensing even if it presents some limitation regarding the suitable film thickness for SPR excitation. Moreover, the response and recovery times during the anal5fle/sensing layer interaction appears shortest in the case of optical absorption measurements. Further investigations are in... 

In addition, the integration of modem optical technology and electrochemical techniques for sensing applications appears to be a powerful new approach. A new type of optoelectrochemical sensor for chlorine, based on an electrochromic thin-film sensing layer placed on top of a planar waveguide, has demonstrated the applicability of this combined approach. 

Figure 6. Optical fibre biosensor setup, (a) Optical fibre bundle (b) thermostated reaction vessel (c) reaction medium (d) sensing layer (e) stirring bar (f) septum and needle guide for sample injection (g) PVC jacket (h) screw-cap for securing the sensing layer.

Figure 7. (a) Flow diagram of the optical fibre continuous-flow system for bioluminescence and chemiluminescence measurements S, sample C, carrier stream PP, peristaltic pump IV, injection valve W, waste FO, optical fibre FC, flow-cell, (b) Details of the optical fibre biosensor/flow-cell interface a, optical fibre b, sensing layer c, light-tight flow-cell d, stirring bar. 

Figure 8. Flow cell for electrochemilumiiiescence measurements (a) glassy carbon electrode (b) sensing layer (c) reagent solution outlet (d) Plexiglas window (e) liquid core single optical fiber (f) stirring bar (g) reagent solution inlet (h) platinum electrode.

A flow injection optical fibre biosensor for choline was also developed55. Choline oxidase (ChOX) was immobilized by physical entrapment in a photo-cross-linkable poly(vinyl alcohol) polymer (PVA-SbQ) after adsorption on weak anion-exchanger beads (DEAE-Sepharose). In this way, the sensing layer was directly created at the surface of the working glassy carbon electrode. The optimization of the reaction conditions and of the physicochemical parameters influencing the FIA biosensor response allows the measurement of choline concentration with a detection limit of 10 pmol. The DEAE-based system also exhibited a good operational stability since 160 repeated measurements of 3 nmol of choline could be performed with a variation coefficient of 4.5%. 

Composite Sensing Layer on Glassy Carbon Electrode... 

A tri-enzymatic sensing layer based on kinase-oxidase activities for the detection of acetate was also described. A reaction sequence using acetate kinase, pyruvate kinase and pyruvate oxidase enabled the production of H2O2 in response to acetate injection in the range 10 pM - 100 mM59. 

Based on IgG-bearing beads, a chemiluminescent immuno-biochip has been also realized for the model detection of human IgG. Biotin-labeled antihuman IgG were used in a competitive assay, in conjunction with peroxidase labelled streptavidin59. In that case, the planar glassy carbon electrode served only as a support for the sensing layer since the light signal came from the biocatalytic activity of horseradish peroxidase. Free antigen could then be detected with a detection limit of 25 pg (108 molecules) and up to 15 ng. 

Composite sensing layers, consisting of bioactive molecule-charged beads entrapped in a polymeric structure, have been successfully used to realize multi-purpose biochips for DNA, proteins or enzymes. For all these different biochips, the chemiluminescence and electro-chemiluminescence measurements required only a CCD camera and neither light sources nor optical filters are needed. 

Michel P.E., Gautier S.M., Blum L.J., Effect of compartmentalization of the sensing layer on the sensitivity of a multienzyme-based bioluminescent sensor for L-lactate, Anal. Lett. 1996 29 (7) 1139-1155. 

The absorption-based platforms described previously employed evanescent wave interrogation of a thin sensing layer coated onto a planar waveguide. A sensitivity enhancement strategy for optical absorption-based sensors based on planar, multimode waveguides was developed recently by us18. The objective was to apply this theory to the development of low-cost, robust and potentially mass-producible sensor platforms and the following section outlines the assumptions and predictions of this theoretical model. 

Figure 6 illustrates the platform under consideration in this analysis. The principle of sensor operation is as described previously for absorption-based optical sensors employing evanescent wave interrogation of the sensing layer. 

Figure 7. Sensitivity plotted as a function of angle of incidence on the sensing layer.

The results presented in the previous section were verified experimentally using the setup illustrated in Figure 8 to record the reflectivity of a sol-gel sensing layer as a function of incident angle. The results are plotted in Figure... 

Some important factors that must be considered in the selection of the support for covalent binding are its capacity to bind the enzyme, as the linearity and the limit of detection of the sensing layers will be influenced by this value the mechanical and chemical stability of the support the efficiency of interaction with the analyte or the sample matrix the ease of preparation and the cost, regenerability and availability of the material. 

Rella R., Rizzo A., Licciulli A., Siciliano P., Troisi L., Valli L., Tests in controlled atmosphere on new oprical gas sensing layers based on Ti02/metal-phtalocyanines hybrid system, Materials Science and Engineering C 2002 22 439-443. 

There are various techniques for the immobilization of proteins onto solid supports. To estimate their suitability for preparation of antibody sensing layers one have to consider some general requirements for the sensor function and application. 

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