Detector

BIOMOLECULE (RECEPTOR)

FIGURE 1.1 Principle of operation of a typical biosensor (Byfield and Abuknesha, 1994). Reprinted from Biosensors and Bioelectronics, vol 9, M. P. Byfield and R. A. Abuknesha, pp. 373400, 1994, with permission of Elsevier Science.

a drug dispenser. Such a feedback system could be incorporated into, for example, an artificial pancreas. Thus, two types of biosensors could be used: in vitro and in vivo.

Byfield and Abuknesha (1994) have clearly identified the biological components that may be used as receptors. These include cofactors, antibodies, receptors, enzymes, enzyme systems, membranes, organelles, cells tissues, and organisms. As the number of biosensor applications increases (which it will, due to the need for rapid, reliable, reproducible, accurate, and sensitive analyses) the types of receptors that may be used in biosensors will increase. These same authors have also identified the different types of transducers that may be used. These include optical (fluorescence, absorbance), electrochemical (amperometric, potentiometric, conducti-metric), piezoelectric, calorimetric, acoustic, and mechanical.

Let us illustrate the flexibility of a typical biosensor—for example, an enzyme biosensor—by using three different transduction elements. The glucose-glucose oxidase (analyte-receptor) biosensor is a good example of an electrochemical transduction wherein the analyte is converted to an electroactive product (Byfield and Abuknesha, 1994). Similarly, the lactate-lactate monoxygenase biosensor is a good example of an optical transduction wherein the optical properties of the enzyme are changed upon reacting with the analyte. Finally, the glucose-glucose oxidase biosensor may also have a calorimetric transduction wherein the analyte reacting with the enzyme gives off heat energy.

Among the various properties of a biosensor to be considered in the design—such as specificity, sensitivity, reproducibility, stability, regener-ability, and response time—the two most important are specificity and sensitivity. Alvarez-Icaza and Bilitewski (1993) emphasize that due to the specificity of the biosensor it may be used in complex media such as blood, serum, urine, fermentation broths, and food, often with minimum sample treatment.

A simple example would be of assistance here. Lowe (1985) indicates that the concentration of certain proteins in blood serum may be as low as a few /¿g/L or less as compared to a total protein concentration of 70 g/L. This requires a discrimination ratio of 107-108 to specifically estimate the desired protein. Since other chemicals will also be present in the blood, an even higher discrimination ratio will be required. Lowe defines sensitivity as the ability of a biosensor to discriminate between the desired analyte and a host of potential contaminants. There are two ways to further delineate sensitivity: the smallest concentration of analyte that a biosensor can detect or the degree of discrimination between measurements at any level.

Lukosz (1991) indicates that optics provide the high sensitivity in, for example, integrated optical (IO) and surface plasmon resonance (SPR) biosensors. However, it is the biochemistry that provides the specificity of biosensors. Lukosz adds that the chemoreceptive coating on IO or SP sensors makes them biosensors. In this text we will cover different types of biosensors but we will emphasize and illustrate most of the concepts using antigen-antibody binding. By focusing on one type of example, we can develop it in detail with the hope that similar development is possible for other types of biosensors. Such further development is essential since, in spite of the vast literature available on biosensors and the increasing funding in this area, there are but a handful of commercially available biosensors (Paddle, 1996). This underscores the inherent difficulties present in these types of systems. However, due to their increasing number of applications and their potential of providing a rapid and accurate analysis of different analytes, worldwide research in this area is bound to continue at an accelerated pace.

The transduction of the biochemical signal to the electrical signal is often a critical step wherein a large fraction of the signal loss (for example, fluorescence by quenching) may occur (Sadana and Vo-Dinh, 1995). This leads to deleterious effects on the sensitivity and the selectivity of the biosensor, besides decreasing the quality of the reproducibility of the biosensor. The sensing principles for biosensors may be extended to a chemical sensor (Alarie and Vo-Dinh, 1991). Here the analyte molecule to be detected is sequestered in /3-cyclodextrin molecules immobilized on the distal kv k2 DUAL-STEP INTERACTION

FIGURE 1.2 Truncated-cone structure of ¡3-cyclodextrins exhibiting a hydrophobic cavity (Sadana and Vo-Dinh, 1995).

Reprinted from Talanta, vol 42, A. Sadana and T. Vo-Dinh, pp. 1567-1574, 1995, with permission from Elsevier Science.

FIGURE 1.2 Truncated-cone structure of ¡3-cyclodextrins exhibiting a hydrophobic cavity (Sadana and Vo-Dinh, 1995).

Reprinted from Talanta, vol 42, A. Sadana and T. Vo-Dinh, pp. 1567-1574, 1995, with permission from Elsevier Science.

end of a fiber-optic chemical sensor. Cyclodextrins are sugar molecules that possess the structure of a truncated cone with a hydrophobic cavity (Fig. 1.2). It is in this hydrophobic cavity that the analyte is complexed and placed in a hydrophobic environment. This leads to fluorescence quenching protection (for example, from water) that can lead to a fluorescence enhancement effect (Alak et al, 1984).

Antibodies frequently have been used for the detection of various analytes due to their high specificity. They have been immobilized on various supports for application in immuno-diagnostic assays. Antibodies may be immobilized on, for example, optical fibers, electrodes, or semiconductor chips (Ogert et al., 1992; Rosen and Rishpon, 1989; Jimbo and Saiti, 1988). Lu et al. (1996) indicate that different immobilization chemistries and strategies have been utilized. Linkages to solid surfaces are frequently made by glutaraldehyde, carbodiimide, and other reagents such as succinimide ester, maleinimide, and periodate. However, one has to be very careful during the immobilization procedure as quite a bit of the activity may be destroyed or become unavailable. Some of the factors that decrease the specificity of biosensors include the cross-reactivity of enzymes, nonspecific binding (analyte binding r, r.

Lu, B., Smyth, M. R., and O'Kennedy, R., Analyst, 121, 29-32R (1996). Reproduced by permission of the The Royal Society of Chemistry.

occurs at places where it should not), and interferences at the transducer (Scheller et al, 1991).

Figure 1.3 shows the schematic diagram of an antibody molecule with its fragments (Lu et al, 1996). The Fc fragment comprises the effector functions, such as complement activation, cell membrane receptor interaction, and transplacental transfer (Staines et al, 1993). The F(ab')2 contains two identical Fab' fragments, which are held together by disulfide linkages in the hinge (H) region. The Fab' fragments contain the antigen-binding site. The VH and VL are the variable heavy and light chains, respectively. The CH1 and the CH2 are the constant chains. Note that as the antibody is immobilized on a support, it generally loses some of its activity, as noted in Figure 1.4 wherein some orientations (or conformations) inhibit the formation of the antigen-antibody complex. Lu et al. (1996) emphasize that if the immobilization occurs through the antigen-binding site, then the ability of the antibody on the surface to bind to the antigen in solution may be lost completely, or at least to a high degree. Therefore, one needs to be careful during the immobilization procedure to preserve most, if not all, of the inherent antibody

FIGURE 1.4 IgG antibody configurations on a surface during a random coupling procedure (Lu et al, 1996).

Lu, B., Smyth, M. R., and O'Kennedy, R., Analyst, 121, 29-322 (1996). Reproduced by permission of The Royal Society of Chemistry.

FIGURE 1.4 IgG antibody configurations on a surface during a random coupling procedure (Lu et al, 1996).

Lu, B., Smyth, M. R., and O'Kennedy, R., Analyst, 121, 29-322 (1996). Reproduced by permission of The Royal Society of Chemistry.

activity. Thus, several approaches have been developed to obtain an appropriate orientation of the antibody during the immobilization process. Figure 1.5 shows some of these approaches (Lu et al., 1996).

The antigen-antibody reaction in a typical biosensor has a much higher specificity than the average chemical sensor, and this advantage needs to be

FIGURE 1.5 Oriented antibody immobilization (Lu et ai., 1996). (a) Antibody binds to Fc receptors on surface; (b) antibody is bound to a solid support through an oxidized carbohydrate moiety on its CH2 domain of the Fc fragment; (c) monovalent Fab' fragment bound to insoluble support through a sulfhydryl group in the C-terminal region.

Lu, B., Smyth, M. R., and O'Kennedy, R., Analyst, 121, 29-322 (1996). Reproduced by permission of The Royal Society of Chemistry.

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