Biosensors & Bioelectronics 17 (2002) 147 157 www.elsevier.

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Review

Biosensors in sh production and quality control

V. Venugopal *
Food Technology Di6ision, Bhabha Atomic Research Centre, Mumbai 400 085, India Received 25 April 2001; accepted 15 May 2001

Abstract Fishery products are important not only from a nutritional point of view, but also as an item of international trade and foreign exchange earner for a number of countries in the world. Fish and shellsh are highly perishable, and prone to vast variations in quality due to differences in species, environmental habitats, feeding habits, etc. In addition, they can also function as carriers of several microbial and other health hazards. Therefore, maintenance of quality is of utmost importance in production and trade of shery products. Most of the current quality control techniques are time consuming and cumbersome. There is an excellent scope for the application of biosensors in the seafood industry including the rapidly expanding aquaculture operations for fast assessment of quality. This article discusses the scope of applications biosensors in the seafood industry. 2002 Elsevier Science B.V. All rights reserved.
Keywords: Fish production; Quality control; Biosensors

1. Introduction Fishery products constitute an important item of international trade, currently worth more than US$50 billion, indicating increasing consumer interests in the commodity. During the past few years, global sh production has been around 120 million metric tonnes, which is not sufcient to meet the consumer need. In order to meet the increasing demand, aquaculture of selected sh and shellsh species is growing contributing nearly 20% of total sh availability. Unlike other animal products, quality of sh is more difcult to control due to variations in species, sex, age, habitats and action of autolytic enzymes as well as hydrolytic enzymes of microorganisms on the sh muscle. Aquaculture operations demand precise control of water quality such as pH, biochemical oxygen demand (BOD), salinity, presence of phytoplankton, etc., and use of appropriate feed formulations for optimal yield of the sh species. Possibilities of variations in aquacul* Tel.: + 91-22-5593964; fax: + 91-22-55-05151. E -mail address: venu@magnum.barc.ernet.in (V. Venugopal).

ture practices together with chances of environmental pollution lead to signicant differences in the quality of aquacultured sh. Fishery products have also been recognized as carriers of health hazards such as diseasecausing microorganisms Salmonella sp. and Vibrio sp., in addition to parasites, natural toxins, heavy metals and other pollutants. Presence of high levels ( \ 50 mg per 100 g sh) of histamine has been a cause of food poisoning through consumption of sh such as mackerel. Many of these hazards are particularly severe with respect to aquacultured sh items due to greater chances of environmental pollution of the ponds. The increasing demand in the commodity as well as the need to protect the consumers against food-borne diseases have made regulatory authorities such as the United States Food and Drug Administration and the European Union stipulate stringent guidelines with respect to the quality of processed sh items traded by exporting countries. The need for maintenance of acceptable quality of these products necessitates development of means for precise and rapid quality evaluation. This article deals with the scope of applications of biosensors for evaluation of biochemical spoilage as

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well as assessment of environmental hazards in sh and shellsh.

Since concentrations of ATP, ADP and AMP signicantly change within the rst day of death, Karube et al. (1984) modied the K -value (KI) as: KI = [HxR] + [Hx] [IMP] + [HxR] + [Hx]

2. Biochemical quality changes in shery products Immediately after catch, a number of post-mortem changes are initiated in the sh muscle, which include physico-chemical as well as autolytic reactions causing changes in proteins and lipids. This is followed by biochemical activities of contaminant microorganisms on the sh muscle leading to loss of its freshness. The loss of freshness quality depends on several intrinsic and extrinsic factors such as the nature of the sh, spawning, feeding habits, temperature of the water, method of catch, handling, and storage conditions (Ashie et al., 1996; Connell, 1995; Huis int Veld, 1996; Gill, 1990; Venugopal, 1990). Immediately after death, when respiration ceases, biosynthesis of adenosine triphosphate (ATP) ceases and the nucleotide already present in the muscle undergoes degradation by enzymes present in the muscle, according to the sequence: ATP ADP AMP IMP HxR Hx X U where ADP is adenosine-5%-diphosphate, AMP is adenosine-5%-monophoshate, IMP is inosine-5%monophosphate, HxR is inosine, Hx is hypoxanthine, X is xanthine, and U is uric acid. The initial steps, up to the formation of HxR, proceed relatively fast as a result of endogenous enzymes in sh and shellsh. The oxidation of Hx to X and ultimately to uric acid is much slower and is due to the enzymes from spoilagecausing microorganisms. The enzymes involved in the oxidation of Hx and X and the reactions are as follows: Nucleoside phosphorylase: HxR + Pi Hx + ribose Pi Xanthine oxidase: Xanthine oxidase: Hx + O2 X + H2O2 X + O2 uric acid + H2O2

Several authors have indicated a strong correlation between nucleotide catabolism and loss of freshness (FAO, 1995; Ashie et al., 1996). Because of the ratelimiting step between X and U, HxR and Hx accumulate in the muscle. Based on the concentrations of different nucleotides, the K -value was proposed as an index of freshness by Saito et al. (1959), which was dened as: K -value = [HxR] + [Hx] [ATP] + [ADP] + [AMP] + [IMP] + [HxR] + [Hx]

The K -value has been found suitable for several species, although it has limitation for some sh such as cod (Botta, 1994). Instead of the K -value, a modied kp-value, namely the hypoxanthine/adenine ratio, has also been found suitable for freshness evaluation of certain shellsh (FAO, 1995; Lou, 1998). Of the procedures available, high-performance liquid chromatography (HPLC) affords accurate and reliable results, but is expensive and also time consuming for routine examination in a quality control laboratory. In the post-mortem muscle, the action of spoilage causing microorganisms eventually leads to the accumulation of a number of volatile basic nitrogen (TVBN) compounds including ammonia by the action of bacterial decarboxylases on amino acids and other nitrogenous compounds (Liston, 1980; Alur et al., 1995; FAO, 1995). TVBN content provides information on the terminal spoilage of sh and is suitable for conrmation of sensory data (Antonacopoulos and Vyncke, 1989). Trimethylamine oxide (TMAO) is found in a large number of marine sh and shellsh, which is broken down to trimethylamine (TMA) by the action of the bacterial enzyme, trimethylamine oxide reductase. The presence of signicant amount of trimethylamine, as a component of TVBN compounds, has been related to loss of freshness of marine shery products, since no trimethylamine is found in the rst 3 5 days of their ice storage (Gram and Huss, 1996; Alur et al., 1995). The development of TMA in many sh species is also paralleled by an increase in the content of hypoxanthine. Volatile sulphur compounds such as H2S, CH3SH, (CH3)2S are also typical components of spoiling sh, which are produced by bacterial action on sulphur containing amino acids, cysteine and methionine. Fishery products are highly perishable due to their particular characteristics, as already mentioned. Fish, immediately after catch, needs to be properly chilled to retard autolytic and microbial spoilage (Venugopal, 1990). The normal storage life of shery products chilled immediately after catch ranges from 7 to 15 days depending on the species. It has been observed that a rise in temperature of refrigerated sh from 0 to 3 C doubles the spoilage, while an increase to 10 C can enhance spoilage by a factor of 5 6, signicantly reducing the shelf life. The other processing techniques to control spoilage include freezing, storage under modied atmosphere, dehydration, exposure to low doses of gamma radiation, etc. (Venugopal and Shahidi, 1998).

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3. Current methods for biochemical quality evaluation and their limitations Several subjective and objective methods have been developed for evaluation of quality of shery products. Sensory evaluation of sh by experienced persons is the most widely practised method, where the judges evaluate and interpret reaction to characteristics of food such as appearance, odour, avour and texture, as perceived through senses. However, apart from being expensive, the results can vary depending upon the personal bias of the evaluating panel members, their fatigue, etc. On the other hand, mechanical, instrumental or laboratory methods are objective, easily reproducible and reliable. These include moisture and fat meters, texturometers and computer-aided vision measurement devices for detection of parasites or blood clots in sh llets (Pau and Olafsson, 1997; Connell, 1995). Chemical and biochemical methods entail taking an extract or piece of sh (which may not represent the whole batch of processed sh) and involve laborious techniques extending to several hours. Measurement of TMA content cannot be useful for freshwater sh species, which have negligible amounts of TMAO. Further, TMA does not increase much in concentration during the early stages of spoilage and its analysis requires a good deal of technical skill. TVBN estimation is of somewhat wider application and can be used for products not containing TMA. In order to deliver good quality shery products to the consumers, standards for freshness of the products are necessary.
Table 1 Conventional methods for quality evaluation of shery products Method Sensory evaluation Chemical methods: total volatile basic nitrogen Trimethylamine Ammonia Volatile acids Nucleotide catabolites Histamine H2S, CH3SH, (CH3) 2S Rancidity Reference FAO (1995), Connell (1995)

For example, an amount of 10 15 mg TMA nitrogen, 35 40 mg TVBN or 50 mg hypoxanthine per 100 g meat is usually regarded as the acceptability limits for chilled cod. The values vary depending on the type of sh and the processing technique employed. Dried sh may contain somewhat higher amounts of TVBN (Connell, 1995). Hypoxanthine can be used for more species and products including freshwater species and shellsh, but requires elaborate technique and skill. Measurement of the K -value by HPLC is more complex and time consuming. Microbiological quality evaluation such as estimation of total plate count or colony forming units per gram of sh takes 2 3 days to complete. Although automated systems have been developed to complete the analysis within a few hours, the equipment and skills necessary to use it are relatively costly. The conventional methods of quality evaluation of sh items are summarised in Table 1.

4. Quality problems due to environmental contamination Hazards due to environmental contamination of shery products by pathogenic microorganisms, industrial pollutants, etc., are major problems in the seafood industry. Several pathogenic microorganisms have been recognized to jeopardise the safety of shery products (WHO, 1999; Venugopal et al., 1999; Levin, 1978). The leading cause of food-borne illness during the past few

Remark

Instrumental methods

Microbiological methods

Depends on sight, smell, taste, touch and hearing as judged by experienced persons Convey microdiffusion method Farber and Ferro, Measurement of trimethylamine, ammonia and (1956), Antonacopoulos and Vyncke (1989) other volatile basic nitrogen by titration Wong and Gill (1987) Useful only for marine sh LeBlanc and Gill (1984) Indicates only advanced spoilage Venugopal et al. (1981) Good correlation with bacterial spoilage Saito et al. (1959). HPLC method is used. Degradation products of ATP. Reliable quality indices of several sh/shellsh, based on K -value Lehane and Olley (2000) Histamine is thermostable and hence cooked sh can be analyzed by uorimetric or HPLC methods Determined by gas chromatography Indicates advanced degree of spoilage Tarladgis et al. (1960), Sinnhuber and Yu (1958) 2-Thiobarbituric acid (TBA) value is a good index of oxidative rancidity in sh lipids. Reasonable correlation with sensory properties FAO (1995); Pau and Olafsson (1997). Measures spoilage based on electrical properties, Commercial pH and texturometers, moisture and pH and vision properties of sh muscle. Presence fat analyzers of parasites and blood clots in sh llets are determined by computer-aided vision techniques Total plate count Indicative of microbial spoilage of fresh sh. Conventional techniques take 4872 h. Rapid techniques are now available

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years was salmonellosis, followed by shigellosis, staphylococcal intoxication and gastroenteritis. Some of the common pathogens found in shery products are Salmonella sp., Staphylococcus aureus, different species of Clostridium botulinum, Bacillus cereus, Campylobac ter jejuni, Escherichia coli O157:H7, Vibrio para haemolyticus, Yersinia enterocolitica, and Listeria monocytogenes. The contamination of sh by these pathogens can occur prior to harvest, during capture, processing, distribution and/or storage. Understanding the profound inuence of factors such as environment, process, and distribution conditions on shery products, a need has been felt for quality assurance and adoption of standards in the sh industry in order to safeguard the health of the consumer. For example, a European Union Directive (91/492) has laid down rules that live bivalves must contain fewer than 300 faecal coliforms or fewer than 230 E. coli per 100 g shellsh esh and contain no Salmonella sp. Marine products imported into Japan should not contain V. cholerae, faecal coliform or Staphylococcus sp. These aspects have been recently discussed by Venugopal et al. (1999). Other environmental hazards that include heavy metals, pesticides and antibiotics may also be present in processed sh, particularly in the case of aquacultured shery products, due to their increased chances of contamination from environments (WHO, 1999). Industrial pollution is responsible for the high content of mercury in sh caught from coastal waters. Regulatory authorities have stipulated that the concentration of mercury in edible portion should not exceed 0.5 1.0 p.p.m. on wet weight basis. Similarly, in the USA, the Food and Drug Administration have set action levels for maximum permitted concentrations, namely, 5.0 mg of the insecticide DDT and its breakdown products, DDE and TDE, 0.30 mg dieldrin and aldrin, 2.0 mg polychlorinated biphenyls. Controls of pond water quality with respect to BOD, salinity level, pH, content of phytoplankton, etc., are also essential for optimal aquaculture operations. Poisoning of humans due to the consumption of sh belonging to the Scombroid family, including mackerel, tuna, herring and marlin, has been attributed to the presence of high levels of histamine and also other biogenic amines including putrescine, cadaverine, spermidine, agmatine, and tyramine. These are generated by the action of bacterial decarboxylases on histidine and related amino acids (Lehane and Olley, 2000). The maximum concentration of histamine permitted by European Union and USA regulations, and in Codex Alimentarius Standards is 20 mg per 100 g sh. Several sh and shellsh may be also sources of toxins such as paralytic shellsh poison, diarrhetic shellsh poisoning, ciguatera, tetrodotoxin, etc., which they imbibe when feeding of certain marine algae. It is essential that products intended for consumption should carry minimum of these toxins (Rawles et al., 1996).

5. Biosensors for biochemical quality evaluation The use of biosensors in foods can be broadly divided into two groups: enzyme sensors for food components, and immunosensors for pathogenic bacteria and pesticides. Their applications in the food industry include proximate analysis, nutritional labelling, determination of pesticide residues, naturally occurring toxins and anti-nutrients, processing changes, microbial contamination, enzymatic inactivation, and BOD of wastes (OConnell et al., 2000; Suleiman and Guilbault, 1994; Wagner and Guilbault, 1994; Luong et al., 1991). These devices, as discussed elsewhere, are analytical devices composed of a biological recognition element (such as an enzyme, antibody, receptor, or microbe) coupled to a chemical or physical transducer. The biological component is normally immobilized at or near the transducer surface in order to maximise the response. The transducers [electrochemical (electrodes), mass (piezoelectric crystals or surface acoustic wave devices), optical (optrodes), or thermal (thermistors or heat-sensitive sensors)] convert the particular biochemical event into an electrical signal, providing a biochemically specic detector that can be easily interfaced to a computer or automated system. Many enzymes and cells consume and/or produce charged species that can be monitored by potentiometric, amperometric, or conductometric techniques. The details of construction of biosensors have been described by several authors (Turner, 2000; Suleiman and Guilbault, 1994; Wagner and Guilbault, 1994; Brooks et al., 1991; Karube and Tamiya, 1987). Signicant developments in biosensors for sh quality measurement started in the 1980s, as recently reviewed by Venugopal et al. (2000). Pioneering work was carried out by Watanabes group to measure nucleotide concentrations to evaluate sh freshness (Watanabe et al., 1983, 1984a,b, 1987). An enzyme sensor specic for Hx in sh was developed using an immobilized xanthine oxidase membrane and an oxygen probe. The enzyme was covalently immobilized on a membrane prepared from cellulose triacetate, 1,8-diamino-4-amino methyl octane using glutaraldehyde. Hx is oxidised to uric acid and hydrogen peroxide by the immobilized enzyme, the output and current of the oxygen probe decreasing due to oxygen consumption. A linear relationship was obtained between current decrease and Hx concentrations in the range 0.06 1.5 mM. The sensor could be used for more than 100 assays and had a storage life of 30 days at 5 C (Watanabe et al., 1983). In another system, both xanthine oxidase and nucleoside phosphorylase were co-immobilized on a membrane already mentioned, for measurement of both Hx and HxR. The enzyme sensor responded to Hx and HxR in the presence of phosphate, while it responded only to Hx in the absence of phosphate. A linear correlation was observed between current decrease and

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the concentration of Hx and HxR in the range 0.5 2.0 mM. A system consisting of nucleotidase, nucleoside phosphorylase and xanthine oxidase could also measure IMP. Measurements with respect to sh such as seabass, mackerel, yellowsh, etc., showed a good correlation when compared with the conventional enzymatic assay (Watanabe et al., 1984a,b). Shen et al. (1996) developed a simpler method that uses a xanthine oxidase electrode. The enzyme was immobilized on a silk membrane, onto which a platinum disc and copper wire were attached. This device was highly sensitive to Hx levels in sh and could therefore be used to assess sh freshness. Karube et al. (1984) developed an enzyme sensor system consisting of a 5%-nucleotidase membrane and a nucleoside phosphorylase xanthine oxidase membrane attached to an oxygen electrode. A small anion-exchange resin column was connected to the enzyme sensor for separation of nucleotides in order to measure each nucleotide concentration. Good correlation was obtained between K -values determined by the sensor and conventional methods. Another biosensor system has been developed to measure the Hx concentration ratio Hx/(Hx + IMP + HxR). The system consisted of a detection chamber equipped with an amperometric electrode using immobilized xanthine oxidase for measurement of Hx. The sensor detected both hydrogen peroxide and uric acid released during the oxidation of Hx. Immobilized nucleosidase was used for conversion of IMP to HxR. After injection of soluble nucleoside phosphorylase and phosphate, the resulting HxR was introduced for determination of the Hx ratio. The system, which had the exibility for measuring the K -value, was successfully used to measure the quality of several sh varieties (Luong and Male, 1992). A multi-enzyme electrode sensor system has been developed for simultaneous determination of AMP, hypoxanthine, inosine and IMP. These compounds were determined according to the reaction: AMP IMP HxR Hx uric acid where AD is adenosine monophosphate (AMP) deaminase, NT is 5%-nucleotidase, NP is nucleoside phosphorylase, PI is inorganic phosphate, and XO is xanthine oxidase. Enzymes were covalently bound to a membrane prepared from cellulose triacetate, 1,8-diamino-4-amino methyloctane and glutaraldehyde. Sensors for Hx, HxR, IMP were prepared by attaching membrane of XO, XO NP, XO NP NT and XO NP NT and AD, respectively, to four oxygen electrodes. Each sensor was based on the principle that the output current of the oxygen electrode decreased due to oxygen consumption, when hypoxanthine is oxidised to uric acid
AD NT NP, PI XO, O2

by xanthine oxidase. The apparatus, in addition to the multi-electrode sensor, consisted of relay controller and A/D converter. For operation, phosphate buffer solution (0.05 M, pH 7.8) containing 0.1 mM cysteine transferred continuously to the sensor system by a peristaltic pump at a ow rate of 1.0 ml per min. After the output current became steady, a 20 50 ml aliquot of a mixture of hypoxanthine, inosine and IMP or a perchloric acid extract of sh muscle, was injected directly into the ow line which was turned off after 600 s. The data obtained from the sensors were analyzed by the computer and the freshness pattern was displayed on the monitor screen. Each assay took 8 min. The system has been successfully used for quality evaluation of seabream, ounder, abalone, etc. (Watanabe and Turner, 1993; Watanabe et al., 1984c). A simple and rapid method using an ammonia ionselective electrode to measure volatile bases in sh has been proposed by Pivarnik and Thiam (1998). The probe could also be used to measure TMA. Storage trials on eight sh species illustrated a correlation with the method that reected nitrogen concentration based on total volatile base analysis. Gamati et al. (1991) used immobilized cells of Pseudomonas amino6orans to measure trimethylamine. The amine could be measured within a range of (5 26) 10 3 mmol with a response of 3.5 min. Urea is present in sh belonging to the Elasmobranchs, and this is degraded to ammonia during spoilage. Amperometric enzyme or microbial probes for ammonium and urea, which have better sensitivities as compared with potentiometric devices, have been developed using immobilized glutamate dehydrogenase and urease enzymes coupled with platinum electrodes. These devices could measure ammonium and urea up to 0.2 mM (Betrocchi et al., 1996; Sheppard et al., 1996; Pandey and Pandey, 1991; Riedel et al., 1990). They may also be employed for evaluation of squid quality, for which formation of ammonia is a good index of quality loss (LeBlanc and Gill, 1984). Fibre-optic biosensors for urea are also available (Schaffar, 1994). Rhines and Arnold (1995) reported a sensor using urease bound to carboxyuorescein dye for uorescence detection, which can measure 0.05 2.5 mM urea with a response time of 1.3 7 min. However, it has a stability of only one day. In contrast, the device reported by Wolfbeis and Li (1992) has stability of more than one month and can detect urea within a range 0.03 0.6 mM with a response time of 4 min. Sensors have been developed for histamine and other polyamines. A polyamine biosensor has been developed using putrescine oxidase immobilized on microplanar thin lm hydrogen peroxide electrodes. The enzyme oxidized putrescine along with cadaverine, spermidine, agmatine, and tyramine to give the respective aldehydes. The consumption of oxygen during the oxidation process was measured by an oxygen electrode,

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Table 2 Enzyme-based biosensors for sh freshness evaluation Enzyme Xanthine oxidase Transducer O2 electrode Metabolite Hypoxanthine Inosine, hypoxanthine K -value (see text); inosine (HxR) K -value (see text) Hypoxanthine KI-value Fish Tuna, seabream, yellowtail Tuna, seabream, yellowtail Rainbow trout, haddock, carp, sole Detection range 0.061.5 mM 0.52 mM Reference Watanabe et al. (1983) Watanabe et al. (1984b) Mulchandani et al. (1990)

O2 electrode Xanthine oxidase, nucleoside phosphorylase Nucleoside phosohorylase, Amperometric xanthine oxidase electrode Immobilised bacterial cells Xanthine oxidase 5%-Nucleotidase, nucleoside phosphorylase, xanthine oxidase O2 electrode Amperometric electrode O2 electrode

K -value (01); HxR (3.6143 mM) Bluen, tuna, yellowtail 526 mM

Fish Seabass, mackerel, ounder Cod, tuna, haddock, perch, ounder 00.5 mM

Watanabe et al. (1987), Hoshi et al. (1990) Shen et al. (1996) Karube et al. (1984)

Ammonia electrode Xanthine oxidase, nucleoside phoshorylase Nucleotidase Pyruvate oxidase, octopine dehydrogenase Diamine oxidase

Ammonia, TMA

1060/530 mg ammonia/(TMA)

Pivarnik and Thiam (1998)

Amperometric electrode O2 electrode Amperometric electrode Amperometric electrode Oxygen electrode

Putrescine oxidase Sulphite oxidase

Sockeye salmon, Pacic cod, Herring Octopine and other Scallop 040 mM amines octopine Histidine, Sole, rainbow trout 25 mM6 mM Cadaverine, Putrescine Polyamines Pollack 0.033 mM Sulphite Different sh 5550 mM

K -value (see text)

Luong and Male (1992) Shin et al. (1998) Male et al. (1996)

Chemnitius et al. (1992) Mulchandani et al. (1991)

which gave a linear response between putrescine oxidation and oxygen consumption within a range of 0.03 3 mM amine. The sensor also responded linearly, when used in sh extracts (Chemnitius et al., 1992). Male et al. (1996) used puried diamine oxidase from porcine kidney immobilized onto a nylon membrane that was attached to an amperometric electrode. The biosensor was linear up to 6 mM with a limit of 25 mM for histamine, putrescine, and cadavarine. The enzyme membrane was stable for 2 months at 5 C and can be used for 60 assays. Formation of octopine from arginine and pyruvic acid is one of the major biochemical processes occurring in scallop muscle post-mortem. About 1% (w/w) octopine is accumulated during a 5-day storage of the scallop in ice. An octopine sensor for quality assessment of scallop freshness developed by Shin et al. (1998) is based on the immobilised enzymes octopine dehydrogenase and pyruvate oxidase. During the reaction, octopine is oxidized to pyruvic acid by the dehydrogenase enzyme; the pyruvic acid generated is further oxidized by pyruvate oxidase to acetyl phosphate. In addition to oxygen electrode used to measure oxygen consumption, the sensor included a reactor and ow cell. A good correlation was obtained between the

octopine content in scallop adductor muscle as determined by the sensor and a HPLC method. Apart from nitrogen metabolites, biosensors are available for lipid derivatives. An amperometric glyceride biosensor has been developed for determination of glycerol and triglycerides using glycerol dehydrogenase (GDH) and lipase. Oxidation of glycerol by glycerol dehydrogenase produces NADH, the electrochemical oxidation of which is measured with a saturated Ag/ AgCl electrode. GDH is immobilized on the surface of a carbon electrode. Sensitivity of the electrode varied from 2 to 9 mM (Laurindavicius et al., 1996). Table 2 summarises the various enzyme-based biosensors developed for measurement of sh quality.

6. Biosensors for monitoring environmental hazards

6.1. Biosensors for pathogenic microorganisms

The microbial contents of shery products including pathogens and their toxins are important indicators of environmental pollution, safety and consumer acceptability, as already pointed out. Rapid information on any microbial health hazard is essential for fast move-

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ment of the commodity in the consumer markets. Immunosensors are more applicable to the area of food safety. The possibility to use antigens in combination with a transducer such as a piezoelectric crystal detector offers the ability to develop a group-specic piezo-immunosensor for the detection of an entire family of bacteria and also insecticides. The technique does not require the use of a label as in the case of radio-immunoassay techniques. Ivintski et al. (1999) have reviewed the different techniques used for bacteriological detection using immunosensors. Whereas an enzyme-linked immunsorbent assay in many cases may satisfy the needs of food analytical laboratories, biosensors based on the use of PZ crystals may nd applications in semicontinuous control as well (Guilbault and Luong, 1991). A reasonably good signal was observed upon exposure of piezoelectric crystals coated with Salmonella antibody through polyethylenimine. The sensor allows detection of 106 109 cells of the bacterium per millilitre (Prusak-Sochaczewski et al., 1990). Crowley et al. (1999) developed a rapid immunosensor for L. monocy togenes in milk, based on amperometric detection, which could be performed in 3.5 h. Rapid and reliable screening procedures for enterobacterial (including Salmonella typhimurium, Shigella dysenteriae, E. coli, Proteus, Serratia and Klebsiella ) contamination of food including shery items and drinking water have been developed using antibodies raised against enterobacterial common antigen, a glycophospholipid of the outer bacteria membrane. Plomer et al. (1992) described an immunosensor for the detection of all enterobacteria in food and drinking water, using monoclonal antibodies against the enterobacterial common antigen. Rasooly and Rasooly (1999) have reported a biosensor that can measure staphylococcal enterotoxin A in food within 4 min. Abdel-Hamid et al. (1999) developed a sensor for E. coli 0157:H7 with a limit of detection of 100 cells per ml. Table 3 indicates some of the major environmental hazards of shery products and biosensors available for their detection. DNA hybridization is another rapid technique that offers the specicity and sensitivity required for the detection of microorganisms in foods, which could be integrated into biosensors. The extent of hybridization between the specic DNA probe and the searched-for DNA could be coupled with the use of uorescence, bioluminescence, electrical detection (including piezoelectric method) and antibody-enzyme-based methods, and is useful for the detection of pathogens (Sharif and Prasad, 2000; Whitaker, 1994; Jones, 1991).

clams. Biosensors for detection of pesticides have been reported (Dankwardt and Hock, 1997; Ivanov et al., 2000). Due to their small size, the antibodies are raised against pesticides conjugated to immunogenic carriers. This increases the difculty in producing immunosensors. Bender and Sadik (1998) produced an electrochemical immunosensor to detect pesticides. A highly sensitive technique for the determination of mercury, silver and copper in the p.p.b. range based on the inhibition of urease has been developed (Danielsson and Mosbach, 1998). Detection of antibiotics in cultured sh is very important. Penicillins in the range 5 30 mmol can be detected using immobilized E. coli with a response time of 8 min (Galindo et al., 1990). Cephalosphorins can be estimated with the range 50 100 mg per ml using Citrobacter freundii, with a response time as low as 10 s (Matsumoto et al., 1979).

7. Potentials of biosensors for aquaculture operations Cultivation of sh species by aquaculture (freshwater, estuarine or marine environments) requires control of salinity, BOD, pH, phytoplankton levels and temperature of pond water and feeding the juveniles with good quality aquafeeds. BOD is widely used as an indicator of the amount of biodegradable organic compounds in waste water, which needs to be properly controlled for optimal growth of sh. Unhygienic aquafeeds, particularly those prepared from sh meal, have been a major

Table 3 Major environmntal hazards in shery products and some examples of biosensors for their estimations Environmental hazard Microbial pathogens Salmonella sp. Shigella sp. Vibrio sp. Escherichia coli Vibrio cholerae Vibrio parahaemolyticus Aeromonas hydrophila Listeria monocytogenes Yersinia enterocolitica Clostridium botulinum Staphylococcus aureus Heavy metals Mercury, copper, silver Pesticides Polychlorinated biphenyls Biosensor

Prusak-Sochaczewski et al. (1990) Ivintski et al. (1999) Crowley et al. (1999) Plomar et al. (1992) Abdel-Hamid et al. (1999) Rasooly and Rasooly (1999) Dmitri et al. (2000)

Nedelkor et al. (2000) Danielsson and Mosbach (1988) Ivanov et al. (2000) Bender and Sadik (1998) Dankwardt and Hock (1997)

6.2. Biosensors for pesticides, hea6y metals and antibiotics in sh

Polychlorinated biphenyls and other pesticides are found throughout the food chain including sh and

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V. Venugopal / Biosensors & Bioelectronics 17 (2002) 147 157 Table 4 Some potential areas of applications of biosensors in aquaculture operations Antibiotics and other antimicrobials Biochemical oxygen demand (BOD) Insecticides Health care of cultured sh, e.g. lactic dehydrogenase activity in body uids Heavy metals (mercury, cadmium, etc.) Herbicides Microbial toxins Nitrate, nitrite Pathogenic microorganisms (Enterobacteria) Polyamines (histamine) Salinity Sulphides

source of pathogenic bacteria in farms and farm-raised sh (Kume et al., 1983). Fish and shellsh produced from freshwater and coastal aquaculture are also more prone to environmental contamination by chemical and biological agents. Such hazards also vary depending on the methods of farming sh, ranging from intensive commercial operations to small-scale systems, management practices and environments (WHO, 1999; Venugopal et al., 2001). Food borne trematode infections and food-borne diseases associated with pathogenic bacteria, residues of agrochemicals, veterinary drugs, and heavy metal contamination have all been identied as health hazards. Conventionally, BOD tests take 5 days to complete and as a consequence are unsuitable for direct process control. Several biosensors have been developed for BOD measurements using microbial cells such as Pseu domonas sp., Bacillus sp., and some thermophilic bacteria. These systems can measure BOD values as low as 1 mg per litre within a maximum of 30 min. Recently, an algal biosensor for monitoring water toxicity in estuarine environments has been proposed. The sensor was obtained by coupling the alga Spirulina subsalsa to an amperometric gas diffusion electrode. The analytical device allows the monitoring of the evolution of phytosynthetic oxygen and the detection of alterations due to toxic effects caused by environmental pollutants (heavy metals, triazinic herbicides, carbamate insecticides). In all the cases, a dose-related inhibition of phytosynthetic activity with good reproducibility could be detected (Campanella et al., 2000; Frense et al., 1998). Biosensors for evaluation of the quality of refrigerated and frozen aquacultured seabass reared in aerated and hyperoxic conditions have been reported (Poli et al., 2000). Nanto et al. (1993) reported an aluminium-dopex zinc oxide thin lm gas sensor capable of detecting freshness of seafoods. Aquacultured sh may pose bacterial hazards as a result of contamination of water through sewage or through unhygienic feeds (Ranjit, 2000). The pathogenic bacteria include Salmonella sp., E. coli O157:H7, Campylobacter sp. and Vibrio sp. Sensors have been developed for detection of these pathogens (Ranjit, 2000; Pathirana et al., 2000; Plomer et al., 1992). DNA hybridization probe techniques with the required biosensor systems are available commercially (Whitaker, 1994). Biosensors for quality evaluation of refrigerated and frozen sea bass reared in aerated or hyperoxic conditions have been reported recently by Poli et al. (2000). As mentioned earlier, potential hazards in aquacultured sh are generally due to environmental pollution. Presence of residues of antimicrobial drugs in farmed shrimp has been increasingly reported in recent years. The problem of drug residues such as oxolinic acid and oxytetracycline in cultured shrimp in the Far East has

had a profound effect on acceptability of their products by countries such as Japan and USA (Weston, 1996). Biotechnological diagnostic tools for disease control and prevention in aquaculture have been developed. These include monoclonal antibodies, polymerase chain reaction, enzyme-linked immunoassays, latex agglutination tests, DNA nger printing and probes. These were discussed recently by Sharif and Prasad (2000). Cultivated sh, generally, become stressed due to restricted living space. Injury due to collisions and illness result from the environment becoming polluted with excrement. Therefore, health care of cultivated sh is very important. Determination of various enzyme activities in the body uids is an important diagnostic tool for health care of the sh. A biosensor for continuous ow analysis of lactic dehydrogenase (LDH) has been developed for the purpose. The sensor system consisting of immobilized pyruvate oxidase membrane, oxygen electrode and ow cell, measured oxygen consumption during oxidation of lactic acid by LDH to pyruvic acid, which is further oxidised by pyruvic oxidase associated with oxygen consumption (Okuma et al., 1989). Table 4 summarises potential applications of biosensors in aquaculture.

8. Commercial biosensors Despite the extensive research performed in the past two decades and the clear demand for on-line measurement, biosensors have yet to be widely accepted for industrial bioprocess monitoring (Scheller et al., 1985; Brooks et al., 1991; Luong et al., 1991). The most successful biosensors available commercially are disposable glucose sensors for testing blood sugar of diabetic patients. Boujtita et al. (1999) reported their efforts on making biosensors applicable to industrial needs. They compared different modes of production of renewable carbon paste electrodes and claimed that they could produce high analytical quality sensors. Cho et al.

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(1995) developed a new device for the rapid measurement of quality of wet sh using a microcomputer attached to a sensor. The portable device could rapidly assess changes in quality of several sh stored under varying temperatures in terms of indices such as the K -value, TVBN, etc., suggesting its potential as a quality indicator kit for sh markets. However, such devices are yet to be commercialised. Commercial biosensors for measuring biochemical oxygen demand are available, and these have signicant application in aquaculture. A collaborative project among six research institutions and a German company is underway to examine the commercial production of biosensors for a variety of applications in Europe (Gibson, 1998). Coupled enzyme systems, known as the KV-101 Freshness meter and Microfresh are available for industrial use (FAO, 1995). A comparable amperometric biosensor system (platinum versus silver/silver chloride at + 0.7 V), designed for the determination of sh freshness, is made by Pegasus Biotechnology, Canada. In this device, immobilized nucleotidase, nucleoside phosphorylase, and xanthine oxidase convert the degradation products of ATP into uric acid and hydrogen peroxide (Mulchandani et al., 1990). Similarly, the Oriental Electric Co, Japan, markets the KV-101 freshness meter, a system containing soluble enzymes together with an oxygen electrode (Luong et al., 1991). Using this instrument, K -values were measured for sh species such as horse mackerel, sardine and Pacic herring purchased from department stores, supermarkets and smaller retail stores (Nomoto and Ohno, 1987). An ammonia ion-selective electrode has the potential to replace conventional methods for volatile basic nitrogen measurement for rapid on-site screening of sh quality (Pivarnik and Thiam, 1998). Test kits for ammonia based on glutamate dehydrogenase are now available from Sigma, USA, and Boehringer Mannheim, Germany. Commercial enzyme kits are also available for estimation of ethanol formed during sh spoilage (FAO, 1995). In India, seafood processing is one of the major industries earning more than US$1 billion through export. Besides, aquaculture of selected species of sh and shellsh is expanding rapidly in the country. However, the industry has been facing hazards with respect to sh production and trade. The recent widespread diseases in aquaculture have resulted in heavy losses to the farmers. In addition, a few years ago, the presence of pathogens in processed shery products attracted a total ban of export from India from European Union. Although, the ban has been partially lifted, it highlights the need for utmost care in quality of shery items intended for export. Rapid evaluation of quality employing appropriate biosensors can be highly advantageous for the country to streamline sh production,

inland distribution and to adhere to international standards required for processed seafoods.

Acknowledgements The author thanks Dr B.D. Malhotra, National Physical Laboratory, New Delhi, India, for his support.

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