Sample Flow

Absorbent Pad

Sample Capture Control Absorbent Inlet _____Line_Line_Pad

Electrical potential _il_

Reference anode pH probe glass membrane Entrapped blocatalyst Semlpe meable membrane

^pH probe Sample pH probe glass membrane Entrapped blocatalyst Semlpe meable membrane

Figure 13.1 Assay formats: (a) direct; (b) competitive; (c) sandwich; (d) two-colour; (e) lateral flow; (f) quenching/FRET; (g) QCM; (h) REVS; (i) BIAcore/SPR; (j) potentiometric nomenon occurring when polarized light is reflected off a thin metal film under conditions of total internal reflection (Kretschmann, 1971). In the BIAcore system, this thin metal film composed of gold forms the floor of a small flow cell, and can be modified so that antibodies are immobilized on its surface. Running buffer is passed continuously through the flow cell and a sample containing the analyte of interest can be injected into this mobile phase. Any interactions that occur between sample antigen and immobilized antibody results in a change in the local refractive index that subsequently changes the SPR angle (Leonard et al., 2003). The change in intensity of reflected light is plotted against time, producing a sensorgram (Merwe, 2003). BIAcore systems have been used for a range of detection applications, including detection of cancer biomarkers in human saliva (Yang et al., 2005). The underlying principles of this technology are presented in Figure 13.1i.

Amperometric, potentiometric, capacitance and ion-selective field-effect transistor-based sensors. Electrochemical sensors are capable of detecting changes in a solution's electrochemical properties that result from binding or biocatalytical events (D'Orazio, 2003). Electrochemical biosensors are the most common form used for clinical analysis and amperometry is the technique usually applied (D'Orazio, 2003). Amperometric devices exploit electroreactive substances and were demonstrated first in 1962 for the detection of glucose in blood (Clark and Lyons, 1962), which spawned continual progression in their application to this field (Wang, 2001). Potentiometric sensors monitor the electrical potential difference between a reference electrode and an indicator electrode that is placed in the sample solution. The potential difference is related to the concentration of analyte in solution in a logarithmic manner (Leonard et al., 2003). The indicator electrode, although immersed in the sample solution, is surrounded by a semi-permeable membrane coated with an entrapped biocatalyst (see Fig 13.1 j). Changes in capacitance were used in the recent development of a novel formaldehyde-sensitive biosensor (Ben Ali et al., 2006), further expanding the range of electrochemical-based detection techniques. Finally, ion-selective field-effect transistor (ISFET)-based biosensors were recently separated as a distinct class of electrochemical sensors, and have a promising scope for application (Dzyadevych et al., 2006).

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