1.7. Growth of Biosensor

Current Status. Since the development of Clark's glucose sensor, many enzyme electrodes have been developed based on amperometry, potentiometry, and photometry. Some of these biosensors are summarized in Tables 1.6, 1.7, and 1.8. The term 'optode' (see Table 1.8) is used for sensors utilizing optical fiber for light signal transmission. Note that the bioreceptors used are all enzymes (see the 'Bioreceptor' column; enzymes end with suffix '-ase') except the antibody sensor. This represents the current state of the art in biosensor development - that bioreceptors other than enzymes are not explored extensively.

Common Products. The column 'Product detected' represent the type of transducer used.. Note that common products (of biorecognition reactions) are used for measurement. For amperometry, the majority is H2O2 (with the exception of NADH and quinone) which is the common product for oxido-reductase enzymes. For potentiometric biosensors, the majority is acid which can be detected by a pH sensor (CO2 and NH3 are indirectly detected by measuring the change in pH).

Biosensor Configurations. When bioreceptor molecules are combined with a suitable transducer, a biosensor is made. Fig. 1.12 shows various biosensor configurations. Note that the bioreceptor molecules are immobilized in a suitable matrix to form a biolayer which is then placed in the immediate vicinity of a transducer. The transducers ion-selective electrode and FET belong to the potentiometric transducer category; the coated wire belong to the amperometric sensor category; the surface plasma detector and the surface acoustic wave detector belong to the piezo transducer category. The materials of constructions for the transducers are also given in the figure.

Discriminative Membrane. Membranes are one of the essential components of a biosensor. They are used for (1) preventing fouling; (2) eliminating interference; and (3) controlling the operating regime of the biosensor. When a small molecule is the analyte, macromolecules such as proteins can be prevented to enter the active sensing

Table 1.6. Amperometric biosensors

Substrate Bioreceptor Product detected Range, mM
choline choline oxidase H2O2 500
ethanol alcohol oxidase H2O2 0 - 10
formaldehyde f. dehydrogenase NADH 10-3
glucose glucose oxidase H2O2, O2 0-7 g/L
glutamine glutamine oxidase H2O2 0-25
glycerol g. dehydrogenase NADH, O2
hypoxanthine x. oxidase H2O2 4-180
lactate lactate oxidase H2O2 1-40
oligosaccharides glucoamylase, glucose oxidase H2O2 0.1-2.5
phenol polyphenol oxidase quinone

Table 1.7. Potentiometric Biosensors

Substrate Bioreceptor Product detected Range, mM
aspartam L-aspartase NH3 0.1-0.6
fats lipase fatty acids 0.005-0.05
glucose glucose oxidase gluconic acid 0.12-2 g/L
urea urease NH4, CO2 0.01-10
nitrite nitrite reductase NH4 1
penicillin penicillinase H+ 0.2-70
sulfate sulfate oxidase HS
antigen or antibody partner of couple complex 0-100 ppm

Table 1.8. Enzyme sensors based on optodes

Substrate Bioreceptor Product detected Range, mM
ethanol alcohol dehydrogenase NADH 0-1
glucose glucose oxidase O2 0.1-20
urease urease ammonia 0.3-3
lactate lactate monooxygenase pyruvate 0.5-1
penicillin penicillinase penicillinic acid 0.25-10

Table 1.9. Biosensors based on FET (pH)

Substrate Bioreceptor Product detected Range, mM
glucose glucose oxidase gluconic acid 0-20
urea urease CO2, NH3 0-6
penicillin penicillinase penicillic acid 0.2-20
triolein lipase fatty acids 0.6-3

zone by using a membrane that has small pore size. Note that proteins are notorious for causing fouling of the sensor. The transport of charged molecules can be controlled by using ion exchange type membranes. A combination of different discriminative membrane can also be used for blocking the passage of different interferents. A summary is given in Table 1.10.

Sensitivity Requirements. The range and type of analytes are also varied and cannot be considered under a single umbrella. The particular application imposes a final concentration range requirement, but initially the concentration level that must be achieved can be estimated by the type of analyte of interest. Metabolites, for example, are commonly found at a level >10-6 mol/L, whereas hormones may be in the 10-l0-10-5 mol/L range and levels as low as 10-20 mol/L would be desirable. For virus, 10-12 mol/L is desirable. This vast range of concentrations is summarized in Fig. 1.13. It is obvious from this figure that, just based on detection limits, very different approaches should be used for an antigen sensor compared with those measuring ion concentrations.

Fig. 1.12. Various biosensor configurations.

Table 1.10. Discriminative coatings for amperometric biosensors.

Transport mechanism Permeselective film
Size exclusion
Cellulose acetate
Base-hydrolyzed cellulose acetate
Phase-inversion cellulose acetate
Polyaniline, Poly pyrrole
Polyphenol
Gamma radiated poly(acrylonitrile)
Charge exclusion
Nafion
Poly(vinylpyridine)
Poly(ester-sulfonic acid)
Polarity Phospholipid
Mixed control
Cellulose acetate - Nafion
Cellulose acetate - Poly(vinylpyridine)

Fig. 1.13. Detection ranges required for some clinically important analytes.

Immunoassay. An important stream of analytical developments, which has been widely applied, is the immunoassay techniques and the DNA probes. In immunoassay, the binding of antibody and antigen results in an increase in molecular mass and volume. Although current biosensor research is investigating the transduction of this phenomenon, the event is usually followed with a photometric, radioactive or even enzyme marker.

DNA Probe. In DNA probe assay, hybridization of strands of DNA antigen results in an increase in molecular mass and volume. The detection of this event is the same as those of immunoassay. There are many major current reasons for replacing the radioisotopic labels with non-radioactive ones, but the direct use of photometric indicators have rarely provided the same degree of sensitivity, so that enzymes have to date frequently been proven to be the most promising form of labeling. The principle of these label-linked assays is similar for both immunoassays and DNA probes. Both of these techniques are heterogeneous assays - so that they are already developed along the lines of the biosensor concept.

Evolution of Biosensors. Biosensors can be classified into three generations according to the degree of integration of the separate components, i.e. the method of attachment of the biorecognition molecule (= bioreceptor) to the base indicator (transducer) element. In the first generation, the bioreceptor is retained in the vicinity of the base sensor behind a dialysis membrane, while in subsequent generations immobilization is achieved via cross-linking reagents or bifunctional reagents at a suitably modified transducer interface or by incorporation into a polymer matrix at the transduction surface. In the second generation, the individual components remain essentially distinct (e.g. control electronics-electrode-biomolecule), while in the third generation the bioreceptor molecule becomes an integral part of the base sensing element (Fig. 1.14). While these definitions were probably intended for enzyme electrode systems, similar classifications appropriate to biosensors in general can be made. It is in the second and third generations of these families that the major development effort can now be seen.

Fig. 1.14. Three biosensor generations (R: Bioreceptor molecule)

Fig. 1.15. Multidisciplinary nature of bisensor development.