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Figure 4. A generalized biosensor configuration. The biologically sensitive coating comprising an antibody, receptor protein or bio-catalyst, such as an enzyme, organelle, whole cell or tissue slice, generates a physico-chemical change in response to the analyte which is converted into an electrical signal by the transducer, amplified and subsequently processed and outputted. Source: Ref. 3.

interact specifically and reversibly, a change takes place in one or more physico-chemical parameters associated with the interaction, such as a change in proton concentration, release or uptake of gases (02, C02, NH3), specific ions (NH4, monovalent cations, anions), heat, optical density, non-specific ions or electron transfer which if generated in close proximity to a suitable transducer, may be converted into an electrical signal. Specific interactions with the biological material can be exploited in the biosensors for sensing specific food ingredients as well as trace compounds, additives or contaminants, toxins, and marker chemicals indicating the microbial state of the food (28,31,32).

Intimate contact between the biosystem and the transducer is quite important. This is achieved by immobilization of the biological material (biocatalyst) at the device surface, generally by one of several methods. First, by chemically cross-linking the biocatalyst with em inert, generally proteinaceous, material with a bifunctional reagent to form intermolecular bonds between the catalyst and the inert protein. An alternative and commonly employed procedure, physically restrains the biocatalyst at the transducer surface by entrapment in polymer matrices such as polyacrylamide or agarose, or by retention with a polymer membrane comprising cellophane, cellulose acetate/ nitrate, poly(vinyl alcohol) or polyurethane. Finally, a preferred procedure in some cases is to covalently attach the biologically sensitive system directly to the surface of the transducer and thereby achieve intimate contact without incurring the diffusional limitations, sometimes observed with membrane or matrix entrapped systems (30).

The transducer is an electrical device which responds to the products of the biocatalytic process and outputs the response in a form which can subsequently be electrically amplified and displayed. The design of the transducer should accommodate the following features: it must be highly specific for the analyte of interest and respond in an appropriate concentration range; it should have a moderately fast response time, typically 1-60 s; it must be amenable to miniaturization and should ideally compensate internally for adverse environmental effects such as temperature dependency, drift, etc.

Three major types of biosensors currently available are biosensors based on either potentiometric or amperometric transducers (33,34) and those combined with optical fibers

Potentiometric Biosensors

Potentiometric devices operate under equilibrium conditions and measure the accumulation of charge at the electrode surface brought about by some selective process. The best known potentiometric biosensor is based on the ion-selective electrode (ISE) discussed earlier, where an immobilized enzyme is coated over the electrode, making it a potentiometric enzyme electrode. The specificity and sensitivity of various enzymes in catalyzing different reactions are exploited here and the substrates or products are measured as they are produced or consumed at the electrode surface. The glass pH electrode was the first ISE to be used with enzymatic reactions that proceed with the consumption or production of hydrogen ions, although other electrodes have subsequently been utilized. For example, the enzyme urease catalyzes the hydrolysis of urea and may be exploited in the determination of urea by immobilization around a pH, NH4 , HCOf or NH3-gas electrode (30).

The integration of an ion-selective membrane with a solid state FET results in the ion-selective field effect transistor (ISFET) as described under Chemical Sensors. It is possible to immobilize enzymes directly over the gate of an ISFET. The resulting device would be called an enzymat-ically sensitive field effect transistor (ENFET). A major advantage of the ENFET over an equivalent enzyme electrode is its small size. Other advantages of ENFETs are similar to those of ISFETs described earlier. Caras et al. (36-38) reported ENFETs sensitive to glucose and penicillin. In their glucose ENFET (Fig. 5), they had immobilized glucose oxidase enzyme by covalently linking it to an open, polyacrylamide gel matrix and placing it on the gate of a pH-sensitive transistor. The figure shows two pH-sensitive ISFETs on a chip which are identical except one of them has the glucose oxidase enzyme added to its gel while the other does not have any enzyme added. The two ISFETs

Figure 5. Schematic diagram of the ENFET chip sensitive to glucose. One of the gels has glucose oxidase enzyme for glucose sensing. D and S are transistor drain and source, respectively. VG is the applied gate voltage and I is the drain-to-source current. Source: Ref. 38.

were put side-by-side so that the non-specific variations in the transistor outputs which were caused by variations in the ambient temperature, pH, and common noise could be eliminated by differential measurement. Such devices are under development and have future potential for on-line monitoring of glucose and other components in various food processes (39).

Amperometric Biosensors

Amperometric electrodes measure the flux of electroactive species. An example of an amperometric biosensor for food applications is a microbial sensor for detection of fish freshness (40). During storage of fish, larger molecular weight compounds such as proteins and glycogen are gradually degraded into smaller molecular weight compounds, which can be utilized more readily by microorganisms. The sensor (Fig. 6) was prepared by immobilizing spoilage causing bacteria, A. putrefaciens, on a membrane filter. The filter was fixed at the tip of an oxygen electrode and covered with a cellulose acetate membrane. The extent of the assimilation of organic substances (resulting from deterioration of fish during storage) by these immobilized microorganisms can be determined from the respiratory activity of the microorganisms by directly measuring the consumption of oxygen in the oxygen electrode. When extract from a stored fish was allowed to flow over the membranes, oxygen consumption due to the increased respiratory activity of the microorganisms caused a decrease in dissolved oxygen around the microbial membrane and consequently brought a marked decrease in the output current of the oxygen electrode. The current decrease between the initial and the minimum current was used as the measure of fish freshness. The output of the sensor, like many other sensors, is influenced by the number of immobilized living cells, pH, temperature, etc. The fish freshness sensor is one of the few biosensors developed for specific applications to the food industry. A xanthine oxidase-immobilized and carbon-based screen-printed electrode was recently developed for determining fish freshness (41). Amperometric measurements of uric acid and hypoxanthine in fish muscle exudates were effective in calculating if value, the index of fish freshness based on the levels of uric acid and hypoxanthine.

Platinum cathode

Immobilized microbial membrane

Fish extract *

Platinum cathode

Immobilized microbial membrane

Fish extract *

Oxygen electrode {dark type)

Rubber ring

Oxygen permeable Teflon membrane

Figure 6. Schematic diagram of a microbial sensor (an amperometric enzyme electrode) system for fish freshness determination. Source: Ref. 40.

Oxygen electrode {dark type)

Rubber ring

Oxygen permeable Teflon membrane

Cellulose acetate dialysis membrane

Figure 6. Schematic diagram of a microbial sensor (an amperometric enzyme electrode) system for fish freshness determination. Source: Ref. 40.

Another example of an amperometric enzyme electrode is a sensor for rapid in-line determination of lactose and other milk components (42). The sensor was prepared by immobilizing glucose oxidase and yS-galactosidase on a nylon membrane and placing it on the platinum electrode surface. A cellulose acetate membrane was placed between the electrode surface and immobilized enzyme to eliminate electroactive compounds such as ascorbic acid. The layer of immobilized enzymes were covered with a cellulose acetate dialysis membrane in order to prevent microbial attack and leaching of the enzymes. The platinum electrode surface can measure hydrogen peroxide concentration am-perometrically. Lactose from milk entered through the cellulose acetate dialysis membrane and first changed to glucose by the action of /i-galactosidase. The glucose formed reacted with glucose oxidase and formed hydrogen peroxide at the platinum electrode which was reflected by a change in current in the electrode. This method of lactose determination in milk samples does not require any preliminary sample treatment, is inexpensive and also very simple and quick compared to existing titrimetric methods. It is likely to be useful for obtaining rapid analytical data on milk in dairy farms and can be used in-line. More recent applications of this technology include the measurement of aspartame in diet beverages (43), nitrate in drinking water (44), glutamate in seasonings (45), and malate in grape musts and wines (46).

Fiber-Optic Biosensors

Optical fibers are being used for numerous sensing applications (35). The fibers are rugged, more resistant to corrosion than other sensors and immune to electromagnetic interference. The transparent fibers of glass are generally used to guide a light signal to the point of measurement where the light signal changes its parameters in response to physical, chemical, and biological changes at the point of measurement. This modified light signal transmitted back along the fibers is analyzed to derive information about the physical, chemical, and biological changes. Common fiber-optic-based biochemical sensing techniques involve the use of enzymes or substrates immobilized on the tip of an optical fiber for substrates or enzyme activity measurement respectively. Trettank et al. (47) described an optical fiber sensor capable of continuously monitoring glucose. In this feasibility study, glucose oxidase enzyme was physically immobilized in a sensing layer at the end of a fiber optic light guide (Fig. 7). The enzyme catalyzed the oxidation of glucose to give gluconic acid, which in turn, lowered the pH in the microenvironment of the sensor. By following the changes in the fluorescence of a pH sensitive dye incorporated in the sensing layer, the enzymatic reaction could be monitored. The optical isolation layer prevented ambient light or intrinsic sample fluorescence from interfering. The pH sensitive dye used was 1-hydroxypyr-ene-3,6,8-trisulfonate (HPTS) which had an almost linear decrease in bright green fluorescence over the pH 7-6 range. The food industry, with a need for glucose sensing for fermentation monitoring and control, can benefit from such developments. A highly specific and sensitive fluoro-metric fiber-optic biosensor immobilized with glucose-

02 Glu H+ H202 GA

02 Glu H+ H202 GA

Optical fiber

Flu Exc Flu

Figure 7. Cross-section through the sensing layer of the fiberoptic glucose sensor. The directions of the exciting light (Exc) and fluorescence (Flu) are also shown. Source: Ref. 47.

Optical isolation

Sensing layer (Glucose oxidase enzyme and pH-sensitive dye) Polyester foil

Plexiglass

Optical fiber

Flu Exc Flu process monitoring have been put at $59 million. Novel biosensors are currently under development at several institutions around the world. Although much of this development is geared towards biomedical needs, food processing can significantly benefit from them due to the similar types of measurements involved. Future research related to biosensors will focus on immobilization techniques, the matching of biosensitive detection substances to analytes, proper transducing mechanisms, proper packaging of the sensor for use in hostile environments, and the optimization of the sensor response.

Figure 7. Cross-section through the sensing layer of the fiberoptic glucose sensor. The directions of the exciting light (Exc) and fluorescence (Flu) are also shown. Source: Ref. 47.

fructose-oxidoreductase from Zymomonas mobilis was later developed for the dual analysis of glucose and fructose (48).

Another type of fiber-optic-based biological sensor involves bioluminescence, where light is emitted during highly sensitive and specific enzymatic reactions. For example, a bio-luminiscent system (47) for the determination of NADH was produced using the bacterial luciferase and NAD(P)H:FMN oxidoreductase coimmobilized on a polyamide membrane. The bioluminiscent system was maintained in close contact with one end of a optical-fiber bundle. The light intensity obtained during reactions was analyzed using a photomultiplier tube which is a simpler instrumentation as compared to the need for a light source and monochromators in many other types of fiber-optic-based biosensors. The maximum light intensity correlated linearly with the concentration of NADH in this study. Such systems are quite new but hold future promises for composition sensing. An extremely sensitive dual-enzyme fiber-optic biosensor immobilized with glutamate dehydrogenase and glutamate-pyruvate transaminase was reported for measuring glutamate based on reduced NAD luminescence (49). A fiber-optic biosensor immobilized with xanthine oxidase and peroxidase was subsequently reported for assaying the quality of seafood products (50). Krug et al. (51) developed a fiber-optic system to measure total and free cholesterol. Immobilized cholesterol esterase cleaved cholesterol esters, while cholesterol oxidase converted cholesterol to cholest-4-ene-3-one and hydrogen peroxide. Hydrogen peroxide formed a colored complex with a dye that was measured by fiber-optic instruments.

A variety of fiber-optic biosensors have been reported capable of detecting bacterial and mold toxins including Clostridium botulinum toxin A (52), Staphylococcal en-terotoxin B (53), Eschericha coli lipopolysaccharide (54), and the mycotoxin fumonisin B1 (55).

Advantages of biosensors are that they do not require highly technically qualified users in order to generate precise results, they are inexpensive, they generate results in a short period of time thus enabling better control of processes or better information for users, and that they can be used in remote locations. Estimates of possible markets of biosensors in the 1990s for food and other industrial

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