Biosensors & Bioelectronics 17 (2002) 147– 157

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Review

Biosensors in fish 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 shellfish 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 fishery 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 ture practices together with chances of environmental

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 fish
production has been around 120 million metric tonnes,
which is not sufficient to meet the consumer need. In
order to meet the increasing demand, aquaculture of
selected fish and shellfish species is growing contribut-
ing nearly 20% of total fish availability. Unlike other
animal products, quality of fish is more difficult to
control due to variations in species, sex, age, habitats
and action of autolytic enzymes as well as hydrolytic
enzymes of microorganisms on the fish muscle. Aqua-
culture 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 fish 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).
pollution lead to significant differences in the quality of
aquacultured fish. Fishery products have also been
recognized as carriers of health hazards such as disease-
causing 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 fish) of histamine has been a cause of food
poisoning through consumption of fish such as mack-
erel. Many of these hazards are particularly severe with
respect to aquacultured fish 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 dis-
eases have made regulatory authorities such as the
United States Food and Drug Administration and the
European Union stipulate stringent guidelines with re-
spect to the quality of processed fish items traded by
exporting countries. The need for maintenance of ac-
ceptable quality of these products necessitates develop-
ment of means for precise and rapid quality evaluation.
This article deals with the scope of applications of
biosensors for evaluation of biochemical spoilage as

0956-5663/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 9 5 6 - 5 6 6 3 ( 0 1 ) 0 0 1 8 0 - 4
148 V. Venugopal / Biosensors & Bioelectronics 17 (2002) 147–157
well as assessment of environmental hazards in fish
and shellfish.
2. Biochemical quality changes in fishery products
Immediately after catch, a number of post-mortem
changes are initiated in the fish 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 fish 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 fish,
spawning, feeding habits, temperature of the water,
method of catch, handling, and storage conditions
(Ashie et al., 1996; Connell, 1995; Huis in’t Veld,
1996; Gill, 1990; Venugopal, 1990). Immediately after
death, when respiration ceases, biosynthesis of
adenosine triphosphate (ATP) ceases and the nucle-
otide already present in the muscle undergoes degrada-
tion 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 fish and shellfish. The
oxidation of Hx to X and ultimately to uric acid is
much slower and is due to the enzymes from spoilage-
causing microorganisms. The enzymes involved in the
oxidation of Hx and X and the reactions are as fol-
lows:
Nucleoside phosphorylase:
HxR + Pi “ Hx + ribose −Pi
Xanthine oxidase: Hx+O2 “X + H2O2
Xanthine oxidase: 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 rate-
limiting step between X and U, HxR and Hx accumu-
late 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
defined as:
K-value
[HxR] +[Hx]
=
[ATP] + [ADP] + [AMP] + [IMP] +[HxR] + [Hx]
Since concentrations of ATP, ADP and AMP signifi-
cantly change within the first day of death, Karube et
al. (1984) modified the K-value (KI) as:
[HxR] + [Hx]
KI =
[IMP] +[HxR] + [Hx]
The K-value has been found suitable for several spe-
cies, although it has limitation for some fish such as
cod (Botta, 1994). Instead of the K-value, a modified
kp-value, namely the hypoxanthine/adenine ratio, has
also been found suitable for freshness evaluation of
certain shellfish (FAO, 1995; Lou, 1998). Of the proce-
dures available, high-performance liquid chromatogra-
phy (HPLC) affords accurate and reliable results, but
is expensive and also time consuming for routine ex-
amination in a quality control laboratory.
In the post-mortem muscle, the action of spoilage
causing microorganisms eventually leads to the accu-
mulation 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 informa-
tion on the terminal spoilage of fish and is suitable for
confirmation of sensory data (Antonacopoulos and
Vyncke, 1989). Trimethylamine oxide (TMAO) is
found in a large number of marine fish and shellfish,
which is broken down to trimethylamine (TMA) by
the action of the bacterial enzyme, trimethylamine ox-
ide reductase. The presence of significant amount of
trimethylamine, as a component of TVBN compounds,
has been related to loss of freshness of marine fishery
products, since no trimethylamine is found in the first
3–5 days of their ice storage (Gram and Huss, 1996;
Alur et al., 1995). The development of TMA in many
fish species is also paralleled by an increase in the
content of hypoxanthine. Volatile sulphur compounds
such as H2S, CH3SH, (CH3)2S are also typical compo-
nents of spoiling fish, 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 fishery 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 fish from 0 to
3 °C doubles the spoilage, while an increase to 10 °C
can enhance spoilage by a factor of 5–6, significantly
reducing the shelf life. The other processing techniques
to control spoilage include freezing, storage under
modified atmosphere, dehydration, exposure to low
doses of gamma radiation, etc. (Venugopal and
Shahidi, 1998).
 V. Venugopal / Biosensors & Bioelectronics 17 (2002) 147–157 149

3. Current methods for biochemical quality evaluation
and their limitations
Several subjective and objective methods have been
developed for evaluation of quality of fishery products.
Sensory evaluation of fish by experienced persons is the
most widely practised method, where the judges evalu-
ate and interpret reaction to characteristics of food
such as appearance, odour, flavour 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, instrumen-
tal or laboratory methods are objective, easily repro-
ducible and reliable. These include moisture and fat
meters, texturometers and computer-aided vision mea-
surement devices for detection of parasites or blood
clots in fish fillets (Pau and Olafsson, 1997; Connell,
1995). Chemical and biochemical methods entail taking
an extract or piece of fish (which may not represent the
whole batch of processed fish) and involve laborious
techniques extending to several hours.
Measurement of TMA content cannot be useful for
freshwater fish 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 fishery products to the consumers,
standards for freshness of the products are necessary.
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
fish and the processing technique employed. Dried fish
may contain somewhat higher amounts of TVBN (Con-
nell, 1995).
Hypoxanthine can be used for more species and
products including freshwater species and shellfish, but
requires elaborate technique and skill. Measurement of
the K-value by HPLC is more complex and time con-
suming. Microbiological quality evaluation such as esti-
mation of total plate count or colony forming units per
gram of fish 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 fish
items are summarised in Table 1.
4. Quality problems due to environmental
contamination
Hazards due to environmental contamination of fish-
ery 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 fishery products
(WHO, 1999; Venugopal et al., 1999; Levin, 1978). The
leading cause of food-borne illness during the past few

Table 1
Conventional methods for quality evaluation of fishery products

Method Reference Remark

Sensory evaluation FAO (1995), Connell (1995) Depends on sight, smell, taste, touch and hearing
as judged by experienced persons
Chemical methods: total volatile Convey microdiffusion method Farber and Ferro, Measurement of trimethylamine, ammonia and
basic nitrogen (1956), Antonacopoulos and Vyncke (1989) other volatile basic nitrogen by titration
Trimethylamine Wong and Gill (1987) Useful only for marine fish
Ammonia LeBlanc and Gill (1984) Indicates only advanced spoilage
Volatile acids Venugopal et al. (1981) Good correlation with bacterial spoilage
Nucleotide catabolites Saito et al. (1959). HPLC method is used. Degradation products of ATP. Reliable quality
indices of several fish/shellfish, based on K-value
Histamine Lehane and Olley (2000) Histamine is thermostable and hence cooked fish
can be analyzed by fluorimetric or HPLC methods
H2S, CH3SH, (CH3) 2S Determined by gas chromatography Indicates advanced degree of spoilage
Rancidity Tarladgis et al. (1960), Sinnhuber and Yu (1958) 2-Thiobarbituric acid (TBA) value is a good index
of oxidative rancidity in fish lipids. Reasonable
correlation with sensory properties
Instrumental methods FAO (1995); Pau and Olafsson (1997). Measures spoilage based on electrical properties,
Commercial pH and texturometers, moisture and pH and vision properties of fish muscle. Presence
fat analyzers of parasites and blood clots in fish fillets are
determined by computer-aided vision techniques
Microbiological methods Total plate count Indicative of microbial spoilage of fresh fish.
Conventional techniques take 48–72 h. Rapid
techniques are now available
150 V. Venugopal / Biosensors & Bioelectronics 17 (2002) 147–157
years was salmonellosis, followed by shigellosis, staphy-
lococcal intoxication and gastroenteritis. Some of the
common pathogens found in fishery 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 fish by these
pathogens can occur prior to harvest, during capture,
processing, distribution and/or storage. Understanding
the profound influence of factors such as environment,
process, and distribution conditions on fishery prod-
ucts, a need has been felt for quality assurance and
adoption of standards in the fish 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 shellfish
flesh 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 fish, particularly in the case of aquacultured
fishery products, due to their increased chances of
contamination from environments (WHO, 1999). In-
dustrial pollution is responsible for the high content of
mercury in fish 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 fish
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, sper-
midine, 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 Eu-
ropean Union and USA regulations, and in Codex
Alimentarius Standards is 20 mg per 100 g fish. Several
fish and shellfish may be also sources of toxins such as
paralytic shellfish poison, diarrhetic shellfish poisoning,
ciguatera, tetrodotoxin, etc., which they imbibe when
feeding of certain marine algae. It is essential that
products intended for consumption should carry mini-
mum of these toxins (Rawles et al., 1996).
5. Biosensors for biochemical quality evaluation
The use of biosensors in foods can be broadly di-
vided into two groups: enzyme sensors for food compo-
nents, and immunosensors for pathogenic bacteria and
pesticides. Their applications in the food industry in-
clude proximate analysis, nutritional labelling, determi-
nation of pesticide residues, naturally occurring toxins
and anti-nutrients, processing changes, microbial con-
tamination, enzymatic inactivation, and BOD of wastes
(O’Connell 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 com-
ponent is normally immobilized at or near the trans-
ducer 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-sen-
sitive sensors)] convert the particular biochemical event
into an electrical signal, providing a biochemically spe-
cific 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).
Significant developments in biosensors for fish qual-
ity measurement started in the 1980s, as recently re-
viewed by Venugopal et al. (2000). Pioneering work was
carried out by Watanabe’s group to measure nucleotide
concentrations to evaluate fish freshness (Watanabe et
al., 1983, 1984a,b, 1987). An enzyme sensor specific for
Hx in fish was developed using an immobilized xan-
thine 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 rela-
tionship 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 nu-
cleoside phosphorylase were co-immobilized on a mem-
brane 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
 V. Venugopal / Biosensors & Bioelectronics 17 (2002) 147–157
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 fish such as sea-
bass, mackerel, yellowfish, etc., showed a good correla-
tion 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 plat-
inum disc and copper wire were attached. This device
was highly sensitive to Hx levels in fish and could
therefore be used to assess fish freshness. Karube et al.
(1984) developed an enzyme sensor system consisting of
a 5%-nucleotidase membrane and a nucleoside phospho-
rylase–xanthine oxidase membrane attached to an oxy-
gen electrode. A small anion-exchange resin column
was connected to the enzyme sensor for separation of
nucleotides in order to measure each nucleotide concen-
tration. Good correlation was obtained between K-val-
ues 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 immo-
bilized xanthine oxidase for measurement of Hx. The
sensor detected both hydrogen peroxide and uric acid
released during the oxidation of Hx. Immobilized nu-
cleosidase was used for conversion of IMP to HxR.
After injection of soluble nucleoside phosphorylase and
phosphate, the resulting HxR was introduced for deter-
mination of the Hx ratio. The system, which had the
flexibility for measuring the K-value, was successfully
used to measure the quality of several fish 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:
AD NT NP, PI XO, O2
AMP “ IMP “ HxR “ Hx “ uric acid
where AD is adenosine monophosphate (AMP) deami-
nase, NT is 5%-nucleotidase, NP is nucleoside phospho-
rylase, PI is inorganic phosphate, and XO is xanthine
oxidase.
Enzymes were covalently bound to a membrane pre-
pared 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 sen-
sor was based on the principle that the output current
of the oxygen electrode decreased due to oxygen con-
sumption, when hypoxanthine is oxidised to uric acid
151
by xanthine oxidase. The apparatus, in addition to the
multi-electrode sensor, consisted of relay controller and
A/D converter. For operation, phosphate buffer solu-
tion (0.05 M, pH 7.8) containing 0.1 mM cysteine
transferred continuously to the sensor system by a
peristaltic pump at a flow 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 fish muscle, was injected
directly into the flow line which was turned off after
600 s. The data obtained from the sensors were ana-
lyzed 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, flounder, abalone, etc. (Watan-
abe and Turner, 1993; Watanabe et al., 1984c).
A simple and rapid method using an ammonia ion-
selective electrode to measure volatile bases in fish has
been proposed by Pivarnik and Thiam (1998). The
probe could also be used to measure TMA. Storage
trials on eight fish species illustrated a correlation with
the method that reflected nitrogen concentration based
on total volatile base analysis. Gamati et al. (1991) used
immobilized cells of Pseudomonas amino6orans to mea-
sure 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 fish belonging to the Elasmo-
branchs, 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 devel-
oped 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 carboxyfluorescein dye for fluorescence
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 film hydrogen peroxide electrodes. The enzyme
oxidized putrescine along with cadaverine, spermidine,
agmatine, and tyramine to give the respective alde-
hydes. The consumption of oxygen during the oxida-
tion process was measured by an oxygen electrode,
152 V. Venugopal / Biosensors & Bioelectronics 17 (2002) 147–157

Table 2
Enzyme-based biosensors for fish freshness evaluation

Enzyme Transducer Metabolite Fish Detection range Reference

Xanthine oxidase O2 electrode Hypoxanthine Tuna, seabream, 0.06–1.5 mM Watanabe et al. (1983)
yellowtail
Xanthine oxidase, O2 electrode Inosine, Tuna, seabream, 0.5–2 mM Watanabe et al. (1984b)
nucleoside phosphorylase hypoxanthine yellowtail
Nucleoside phosohorylase, Amperometric K-value (see text); Rainbow trout, K-value (0–1); Mulchandani et al. (1990)
xanthine oxidase electrode inosine (HxR) haddock, carp, sole HxR (3.6–143
mM)
Immobilised bacterial cells O2 electrode K-value (see text) Bluefin, tuna, yellowtail 5–26 mM Watanabe et al. (1987),
Hoshi et al. (1990)
Xanthine oxidase Amperometric Hypoxanthine Fish Shen et al. (1996)
electrode
5%-Nucleotidase, nucleoside O2 electrode KI-value Seabass, mackerel, 0–0.5 mM Karube et al. (1984)
phosphorylase, xanthine flounder
oxidase
Ammonia Ammonia, TMA Cod, tuna, haddock, 10–60/5–30 mg Pivarnik and Thiam (1998)
electrode perch, flounder ammonia/(TMA)
Xanthine oxidase,
nucleoside phoshorylase
Nucleotidase Amperometric K-value (see text) Sockeye salmon, Pacific Luong and Male (1992)
electrode cod, Herring
Pyruvate oxidase, octopine O2 electrode Octopine and other Scallop 0–40 mM Shin et al. (1998)
dehydrogenase amines octopine
Diamine oxidase Amperometric Histidine, Sole, rainbow trout 25 mM–6 mM Male et al. (1996)
electrode Cadaverine,
Putrescine
Putrescine oxidase Amperometric Polyamines Pollack 0.03–3 mM Chemnitius et al. (1992)
electrode
Sulphite oxidase Oxygen Sulphite Different fish 5–550 mM Mulchandani et al. (1991)
electrode

which gave a linear response between putrescine oxida-
tion and oxygen consumption within a range of 0.03–3
mM amine. The sensor also responded linearly, when
used in fish extracts (Chemnitius et al., 1992). Male et
al. (1996) used purified 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 occur-
ring 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 assess-
ment of scallop freshness developed by Shin et al.
(1998) is based on the immobilised enzymes octopine
dehydrogenase and pyruvate oxidase. During the reac-
tion, octopine is oxidized to pyruvic acid by the dehy-
drogenase 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 flow
cell. A good correlation was obtained between the
octopine content in scallop adductor muscle as deter-
mined by the sensor and a HPLC method.
Apart from nitrogen metabolites, biosensors are
available for lipid derivatives. An amperometric glyce-
ride 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 devel-
oped for measurement of fish quality.
6. Biosensors for monitoring environmental hazards
6.1. Biosensors for pathogenic microorganisms
The microbial contents of fishery products including
pathogens and their toxins are important indicators of
environmental pollution, safety and consumer accept-
ability, as already pointed out. Rapid information on
any microbial health hazard is essential for fast move-
 V. Venugopal / Biosensors & Bioelectronics 17 (2002) 147–157 153

ment of the commodity in the consumer markets. Im- clams. Biosensors for detection of pesticides have been
munosensors are more applicable to the area of food reported (Dankwardt and Hock, 1997; Ivanov et al.,
safety. The possibility to use antigens in combination with 2000). Due to their small size, the antibodies are raised
a transducer such as a piezoelectric crystal detector offers against pesticides conjugated to immunogenic carriers.
the ability to develop a group-specific piezo-immunosen- This increases the difficulty in producing immunosensors.
sor for the detection of an entire family of bacteria and Bender and Sadik (1998) produced an electrochemical
also insecticides. The technique does not require the use immunosensor to detect pesticides. A highly sensitive
of a label as in the case of radio-immunoassay techniques. technique for the determination of mercury, silver and
Ivintski et al. (1999) have reviewed the different tech- copper in the p.p.b. range based on the inhibition of
niques used for bacteriological detection using im- urease has been developed (Danielsson and Mosbach,
munosensors. Whereas an enzyme-linked immunsorbent
1998).
assay in many cases may satisfy the needs of food
Detection of antibiotics in cultured fish is very impor-
analytical laboratories, biosensors based on the use of PZ
tant. Penicillins in the range 5–30 mmol can be detected
crystals may find applications in semicontinuous control
using immobilized E. coli with a response time of 8 min
as well (Guilbault and Luong, 1991).
A reasonably good signal was observed upon expo- (Galindo et al., 1990). Cephalosphorins can be estimated
sure of piezoelectric crystals coated with Salmonella with the range 50–100 mg per ml using Citrobacter
antibody through polyethylenimine. The sensor allows freundii, with a response time as low as 10 s (Matsumoto
detection of 106 –109 cells of the bacterium per millilitre et al., 1979).
(Prusak-Sochaczewski et al., 1990). Crowley et al.
(1999) developed a rapid immunosensor for L. monocy-
togenes in milk, based on amperometric detection, 7. Potentials of biosensors for aquaculture operations
which could be performed in 3.5 h. Rapid and reliable
screening procedures for enterobacterial (including Cultivation of fish species by aquaculture (freshwa-
Salmonella typhimurium, Shigella dysenteriae, E. coli, ter, estuarine or marine environments) requires control
Proteus, Serratia and Klebsiella) contamination of food of salinity, BOD, pH, phytoplankton levels and temper-
including fishery items and drinking water have been ature of pond water and feeding the juveniles with good
developed using antibodies raised against enterobacte- quality aquafeeds. BOD is widely used as an indicator
rial common antigen, a glycophospholipid of the outer of the amount of biodegradable organic compounds in
bacteria membrane. Plomer et al. (1992) described an waste water, which needs to be properly controlled for
immunosensor for the detection of all enterobacteria in optimal growth of fish. Unhygienic aquafeeds, particu-
food and drinking water, using monoclonal antibodies larly those prepared from fish meal, have been a major
against the enterobacterial common antigen. Rasooly
and Rasooly (1999) have reported a biosensor that can
measure staphylococcal enterotoxin A in food within 4 Table 3
min. Abdel-Hamid et al. (1999) developed a sensor for Major environmntal hazards in fishery products and some examples
E. coli 0157:H7 with a limit of detection of 100 cells per of biosensors for their estimations
ml. Table 3 indicates some of the major environmental
Environmental hazard Biosensor
hazards of fishery products and biosensors available for
their detection. Microbial pathogens
DNA hybridization is another rapid technique that Salmonella sp. Prusak-Sochaczewski et al. (1990)
offers the specificity and sensitivity required for the Shigella sp. Ivintski et al. (1999)
detection of microorganisms in foods, which could be Vibrio sp. Crowley et al. (1999)
Escherichia coli Plomar et al. (1992)
integrated into biosensors. The extent of hybridization Vibrio cholerae Abdel-Hamid et al. (1999)
between the specific DNA probe and the searched-for Vibrio parahaemolyticus
DNA could be coupled with the use of fluorescence, Aeromonas hydrophila Rasooly and Rasooly (1999)
bioluminescence, electrical detection (including Listeria monocytogenes Dmitri et al. (2000)
piezoelectric method) and antibody-enzyme-based Yersinia enterocolitica
Clostridium botulinum
methods, and is useful for the detection of pathogens Staphylococcus aureus Nedelkor et al. (2000)
(Sharif and Prasad, 2000; Whitaker, 1994; Jones, 1991).
Heavy metals
Mercury, copper, silver Danielsson and Mosbach (1988)
6.2. Biosensors for pesticides, hea6y metals and
Pesticides
antibiotics in fish
Polychlorinated biphenyls Ivanov et al. (2000)
Bender and Sadik (1998)
Polychlorinated biphenyls and other pesticides are Dankwardt and Hock (1997)
found throughout the food chain including fish and
154 V. Venugopal / Biosensors & Bioelectronics 17 (2002) 147–157
source of pathogenic bacteria in farms and farm-raised
fish (Kume et al., 1983). Fish and shellfish 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 fish, ranging from intensive
commercial operations to small-scale systems, manage-
ment practices and environments (WHO, 1999;
Venugopal et al., 2001). Food borne trematode infec-
tions and food-borne diseases associated with patho-
genic bacteria, residues of agrochemicals, veterinary
drugs, and heavy metal contamination have all been
identified 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 bacte-
ria. 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 estuar-
ine 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 phy-
tosynthetic oxygen and the detection of alterations due
to toxic effects caused by environmental pollutants
(heavy metals, triazinic herbicides, carbamate insecti-
cides). 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 refrig-
erated and frozen aquacultured seabass reared in aer-
ated and hyperoxic conditions have been reported (Poli
et al., 2000). Nanto et al. (1993) reported an alu-
minium-dopex zinc oxide thin film gas sensor capable
of detecting freshness of seafoods.
Aquacultured fish may pose bacterial hazards as a
result of contamination of water through sewage or
through unhygienic feeds (Ranjit, 2000). The patho-
genic 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 aquacul-
tured fish 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
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 fish, e.g. lactic dehydrogenase activity in
body fluids
Heavy metals (mercury, cadmium, etc.)
Herbicides
Microbial toxins
Nitrate, nitrite
Pathogenic microorganisms (Enterobacteria)
Polyamines (histamine)
Salinity
Sulphides
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 agglutina-
tion tests, DNA finger printing and probes. These were
discussed recently by Sharif and Prasad (2000).
Cultivated fish, 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 fish
is very important. Determination of various enzyme
activities in the body fluids is an important diagnostic
tool for health care of the fish. A biosensor for contin-
uous flow analysis of lactic dehydrogenase (LDH) has
been developed for the purpose. The sensor system
consisting of immobilized pyruvate oxidase membrane,
oxygen electrode and flow cell, measured oxygen con-
sumption during oxidation of lactic acid by LDH to
pyruvic acid, which is further oxidised by pyruvic ox-
idase 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 measure-
ment, 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 dispos-
able 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.
 V. Venugopal / Biosensors & Bioelectronics 17 (2002) 147–157
(1995) developed a new device for the rapid measure-
ment of quality of wet fish using a microcomputer
attached to a sensor. The portable device could rapidly
assess changes in quality of several fish stored under
varying temperatures in terms of indices such as the
K-value, TVBN, etc., suggesting its potential as a qual-
ity indicator kit for fish markets. However, such devices
are yet to be commercialised. Commercial biosensors
for measuring biochemical oxygen demand are avail-
able, and these have significant application in
aquaculture.
A collaborative project among six research institu-
tions 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 me-
ter’ 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 fish freshness, is
made by Pegasus Biotechnology, Canada. In this
device, immobilized nucleotidase, nucleoside phospho-
rylase, and xanthine oxidase convert the degradation
products of ATP into uric acid and hydrogen peroxide
(Mulchandani et al., 1990). Similarly, the Oriental Elec-
tric Co, Japan, markets the KV-101 freshness meter, a
system containing soluble enzymes together with an
oxygen electrode (Luong et al., 1991). Using this instru-
ment, K-values were measured for fish species such as
horse mackerel, sardine and Pacific herring purchased
from department stores, supermarkets and smaller re-
tail stores (Nomoto and Ohno, 1987). An ammonia
ion-selective electrode has the potential to replace con-
ventional methods for volatile basic nitrogen measure-
ment for rapid on-site screening of fish quality
(Pivarnik and Thiam, 1998). Test kits for ammonia
based on glutamate dehydrogenase are now available
from Sigma, USA, and Boehringer Mannheim, Ger-
many. Commercial enzyme kits are also available for
estimation of ethanol formed during fish 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 fish
and shellfish is expanding rapidly in the country. How-
ever, the industry has been facing hazards with respect
to fish 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 fishery 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 fishery items
intended for export. Rapid evaluation of quality em-
ploying appropriate biosensors can be highly advanta-
geous for the country to streamline fish production,
155
inland distribution and to adhere to international stan-
dards 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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