Blosensors: Applications for Dairy Food Industry
EDWARD R. RICHTER
ABSTRACT
Biosensors are defined as indicators
of biological compounds that can be as
simple as temperature-sensitive paint or
as complex as DNA-RNA probes. Food
microbiologists are constantly seeking
rapid and reliable automated systems for
the detection of biological activity. Bio-
sensors provide sensitive, miniaturized
systems that can be used to detect un-
wanted microbial activity or the presence
of a biologically active compound, such
as glucose or a pesticide. Immunodiag-
nostics and enzyme biosensors are two
of the leading technologies that have had
the greatest impact on the food industry.
The use of these two systems has
reduced the time for detection of patho-
gens such as Salmonella to 24 h and has
provided detection of biological com-
pounds such as cholesterol or chymo-
trypsin. The continued development of
biosensor technology will soon make
available "on-line quality control" of
food production, which will not only re-
duce cost of food production but will
also provide greater safety and increased
food quality.
(Key words: biosensor, methods,
microbiology, industrial application)
Abbreviation key: EIA = enzyme imunoas-
say.
INTRODUCTION
The science of biosensors is a multidiscipli-
nary area for which sharp delineation does not
exist (3, 5). A biosensor may be defined in
Received August 19, 1992.
Accepted May 19. 1993.
1993 J Dairy Sci 76:3114-3117
Silliker Laboratories
Columbus, OH 43212
many ways. Two of the most commonly used
definitions of biosensor are given:
1. A biosensor may be a device or instru-
ment comprising a biological sensing
element coupled to a transducer (15). Us-
ing this definition, examples of biologi-
cal sensing elements might include en-
zymes, organelles, antibodies, whole
cells, DNA, and tissue. Transducers
might include those that are electrochem-
ical, calorimetric, optical, acoustical, or
mechanical.
2. A biosensor may be a self-contained ana-
lytical system that responds directly and
selectively to biologically important spe-
cies (5, 10); that is, a device or system
that detects a biological event.
Clearly, the term biosensor is used in di-
verse ways, but, generally, a biosensor should
respond selectively, continuously, rapidly, spe-
cifically, and ideally without added reagent to
biological events. The biosensor does not need
to be an integral part of the transducer. En-
zyme biosensors may respond as quickly as 30
s, but 15 min is acceptable for immunosensors.
In order to define biosensors for use in the
food industry, the types of biological events
that might be acceptable for biosensor meas-
urement need to be known (5).
Food microbiology has changed little in the
past 30 yr. The standard plate count, using
agar-based media in a Petri dish, remains one
of the principle tests used to determine
microbiological quality of food. Recent contri-
butions to science have brought about unique
microbiological and chemical food testing
methodologies. These include advances in
genetic engineering (probe production, assay
development, and amplification polymerase
chain reaction), advances in immunology
(monoclonal antibodies, antibody purification,
immunoassays), electrochemical methods (con-
ductance and impedance), and chemilumines-
cence. All of these methods have the same
3114
 SYMPOSIUM: EVALUATION OF Mll.K AND DAIRY PRODUCTS
objective: to reduce the time required for the
detection of a biological event.
TYPES OF BIOSENSORS
Indicator Organisms
Indicator organisms are 1) microorganisms
or their metabolic products that may indicate
the presence of a pathogen or harmful toxin
(Escherichia coli fecal coliforms, enterococci,
staphylococci, or Pseudomonas aeruginosa); or
2) the possibility that faulty practices occurred
during production, processing, storage, and
distribution (coliform bacteria, Enterobacteria-
ceae, or total Gram-negative bacteria); or 3) the
suitability of a food or ingredient for a desired
purpose (11).
Indicator organisms are used when routine
testing for a multiplicity of pathogens is im-
practical. For some pathogens, reliable
methods are not yet available, making the use
of indicator systems the only way to detect the
potential contamination. Indicator systems are
generally simple to use and economical to use
frequently.
Some problems with the reliability of indi-
cator systems stem from the lack of correlation
between pathogens and indicator systems. In
some instances, the indicator organism may be
part of the normal microbial flora of the food.
Finally, the indicator organism may grow in
the food, which results in the loss of quantita-
tive significance. The problems caused by use
of indicator organisms as indicators of patho-
gens are shown in research describing the
results from microbiological analysis of dried
food samples (14). Of the 5837 samples ana-
lyzed, 1943 contained fecal coliforms, 53 con-
tained Salmonella but no fecal coliforms, and
only 47 contained both Salmonella and fecal
coliforms (14).
Nucleic Acid Probes
In recent years, DNA and RNA probes have
become accepted systems of microorganism
detection. 1be principle of selective detection
is based on the detection of a unique sequence
of nucleic acid bases through hybridization. A
DNA probe is added to DNA or RNA from an
unknown sample. If the probe hybridizes (com-
bines) with the unknown nucleic acid because
3115
of pairing of complementary base recognition,
detection and identification are possible. This
hybridization is usually visualized through
color development combining enzyme im-
munoassay (EIA) technology with the biosys-
tem. Commercially, biosensing nucleic acid
probes exist for the detection of Salmonella.
Listeria. Escherichia coli. and Staphylococcus
aureus.
EIA
Microorganisms are often characterized and
identified by the presence of unique protein-
carbohydrate markers (antigens) located within
the body or the flagella of the cell. Detection
of these unique antigens has been a corner-
stone of diagnostic microbiology for many
years. In recent years, EIA using monoclonal
antibodies have made available rapid and con-
sistent microbiological detection systems. The
most widely used systems employ a sandwich
technique using antibody attached to a polysty-
rene matrix to which the sample is added.
Postincubation, a second antibody, which is
specific for the organism and has been tagged
with an enzyme, is added. The addition of
enzyme substrate to the mixture completes the
EIA. The presence of the specific organism
results in a colorimetric change in the enzyme
substrate, which may be observed visually or
with a spectrophotometer. Most EIA are very
specific but lack sensitivity. Normal sensitivity
is in the range of 106 organisms/mi.
Conductance
When microorganisms metabolize un-
charged substrates, such as carbohydrates. to
intermediates, such as lactic acid, the conduc-
tivity of the medium changes. This change is
measurable by detection of small changes in
the conductivity of the medium between two
electrodes. The amount of charged metabolites
is directly proportional to the growth rate of
the organism and is easily quantifiable. An
adaptation to this technique employs an in-
direct measurement of a microbial metabolite,
C~. When yeast grows, one by-product is
C~. If an electrode is submerged in a base,
such as potassium hydroxide, the absorption of
C~ by the base will change the conductance.
1be amount of change is proportional to the
Journal of Dairy Science Vol. 76, No. 10. 1993
3116
amount of C02 produced by the yeast, which
is proportional to the number of yeast cells or
to the rate of growth of the yeast in the sample.
Bioluminescence
Chemiluminescence is the measurement of
light emitted from a chemical reaction. When
caused by biological enzymatically catalyzed
reaction, this chemical reaction is often re-
ferred to as bioluminescence. One example of
a biosensor system utilizing chemilumines-
cence is the luciferase system. In this system,
ATP from viable microorganisms is detected
and quantified by addition of the sample to the
cofactor luciferin and the enzyme luciferase:
luciferin + luciferase + ATP
-+ AMP + light + C02.
This biosensor is capable of detecting bacteria
in the range of I ()4 cellslml in only a few
minutes.
Enzyme Sensors
Enzyme sensors fall into various classes,
including those that are potentiometric, am-
perometric, electrochemical, optoelectric,
calorimetric, and piezoelectric. Basically, all
enzyme sensors work by immobilization of the
enzyme system onto a transducer. As the sen-
sor comes in contact with the substrate, the
resultant by-product of the enzymatic reaction
produces an electric signal, which is measured
by the transducer. Potentiometric sensors are
the simplest form, composed of an ion-
selective electrode (lSE) in conjunction with a
biological element surrounded by a membrane
(8, 12). Caras and Jamata (4) have developed
an enzyme-based biosensor using penicillinase
immobilized onto the surface of a pH-sensitive
transistor. The biosensor is reported to be able
to detect penicillin in milk as it flows from
truck to dairy. Amperometric sensors measure
current such as the common glucose oxidase
system (2, 12), which is based on the en-
zymatic reaction:
glucose + oxygen + glucose oxidase
-+ H202 + gluconate.
The biosensor can measure either the decrease
in oxygen or the accumulation of H202. Elec-
Journal of Dairy Science Vol. 76. No. 10, 1993
RICHTER
trochemical sensors employ the biological
membrane or immobilized enzyme directly on
a transistor gate (12). As the enzyme catalyzes
a biological reaction, a conductive by-product
is produced, creating current flow across the
gate. Optoelectric sensors use light-emitting
diodes and biological chemicals attached to
fiber optic filaments (12, 13). Recently, the
luciferase gene has been genetically transferred
to biological species, such as phage, that act as
a biological detector for the detection of
specific bacteria. By immobilization of phage
containing the luciferase gene to an optoelec-
tric biosensor, specific bacteria may be de-
tected. Most biological reactions are exother-
mic. A calorimetric biosensor, the thermal
enzyme probe (rEP), employs an enzyme im-
mobilized onto a thermistor (6).
Immunosensora
The high degree of specificity in antigen-
antibody reactions is attractive in the develop-
ment of biosensors. Although the use of
antigen-antibody complex specificity may be
utilized in all types of sensors, recent technol-
ogy has been developed that utilizes mass
changes on a piezoelectric crystal to measure
this specific biological activity (1, 9). The
change in mass that is due to the binding of
biological material, such as antigen to anti-
body, will change the frequency of vibration of
the crystal. This change of vibration may then
be converted to an electrical signal by the
crystal. A rapid Salmonella detection system
utilizing this technology may soon be availa-
ble.
Microbial Sensors
Microbial sensors are defined as microor-
ganisms that are associated with a transducer.
Electrochemical microbial sensors usually de-
tect respiratory activity of the microorganisms.
Given a sample with an unknown concentra-
tion of substrate, this type of biosensor meas-
ures the activity of the microorganism within
the sensor, thus measuring indirectly the sub-
strate concentration. An example of a
microbial biosensor is the ammonia gas sensor
(1). This sensor uses nitrifying bacteria as the
detector immobilized on an oxygen electrode.
As the bacteria oxidize the ammonia, oxygen
is consumed in direct proportion to the amount
of ammonia substrate.
 SYMPOSIUM: EVALUATION OF MILK AND DAIRY PRODUCTS
CONCLUSIONS
Although biosensor research has sporadi-
cally appeared in literature for two decades,
few biosensors are commercially available.
Major drawbacks include the delicate nature of
the biological component and the miniaturiza-
tion of the electrical components. As electronic
innovation continues to deliver smaller and
more reliable electronic circuitry and as biol-
ogy continues to develop the unique under-
standing of enzyme and microbial genetics, the
future will deliver reliable biosensors for the
detection of biological events on-line. The
food industry will benefit from these develop-
ments in the rapid detection of microorganisms
and discrete biological components.
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