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and adsorbed proteins on the substratum surface. For
example, with milk-treated steel, ferrous ions in solution
increased Listeria monocytogenes attachment (36). Ions in
the solution can also act as shields, shielding the surface
charge of the substratum and the bacteria (44) and
increasing bacteria attachment (45, 46). The ionic compo-
sition in the bulk may affect the composition of the
metabolic by products of biofilm cells but not necessarily
affect the physical property of the biofilm (47).
Hydrodynamics. The flow velocity in close proximity to
the substratum and the liquid boundary (hydrodynamics)
has marked influence on the cellular interaction and the
biofilm structure. Cells behave as particles in a liquid, and
the rate of settling and association with a submerged
surface depends greatly on the velocity characteristics of
the liquid (48). After the bacteria has attached, flow rate
or shear force of the liquid affects the biofilm structure
and content (25). More compact, stable, and denser bio-
films were formed at relatively higher hydrodynamic
shear force (49).
Atmosphere. The incubation atmosphere also influ-
ences biofilm formation. For instance, a microaerophilic
and carbon dioxide-rich environment provided a relatively
high rate of biofilm formation, whereas the least amount
of biofilm was formed under anaerobic conditions (43). On
the other hand, anaerobic growth favors maintenance of
mucoid alginate (polysaccharide) production by Pseudo-
monas in cystic fibrosis airways (50).
BIOFILM FORMATION ON THE SURFACE OF THE
PACKAGING MATERIALS
All ‘‘real-life’’ surfaces have substantial nonuniformity,
with the surface irregularities (patterns) size ranging
from nanometers to hundreds of microns. Although a
number of studies have investigated the influence of the
surface topography on biofilm formation by various micro-
organisms, including foodborne pathogens (1, 10, 51–53),
most published results are devoted to the biofilm develop-
ment in flow-through systems. It is difficult to separate
the effects of surface patterns and those of the liquid flow
on bacteria adhesion in such systems.
An ability to adhere to a surface provides an important
survival mechanism for microorganisms (9). The process
of microorganism’s attachment to the material surface is
very complex, and the nature of both the microbial cell
surface and the supporting substrate is important (6). For
example, an electropolished stainless steel substratum
showed significantly lower bacterial cells adhesion rate
and delay in biofilm formation, compared with the sand-
blasted one (54). Planktonic microbial cells are delivered
by diffusion and motility from a bulk medium to the
surface, where a fraction of those cells adheres to the
surface. The dynamics of bacterial adhesion is a unique
characteristic of the specific microorganism; there may
even be differences among the phenotypes and strains of
the same bacterium (7).
Bacterial colonization of surfaces is influenced by two
factors: First, a well-developed surface has higher adsorp-
tion capacity, and therefore the preconditioning organic
film necessary for bacteria attachment is more likely to be
formed on such a surface. On the other hand, surface
topography influences bacterial attachment and prolifera-
tion, limiting the directions of colony growth and limiting
nutrient access.
Surface topography was found to greatly affect the
behavior and morphology of bacterial cells within colonies
during the initial stages of biofilm development. In an
effort to maximize their survival rate, bacteria form
clusters of unique shapes, ranging from two-dimensional
(2-D) single-layer colonies to three-dimensional (3-D) pil-
lar-like structures within grooves. Hence, it is possible to
control initial colony shape by varying the characteristics
of surface constraints. Coupled with surface topography,
starvation may play an important role in the attachment
of bacteria to the surface, which is directly supported by
our observations of bacterial behavior in the surface
confines with limited nutrient access.
In general, surface patterns (i.e., roughness) impact
microbial population in several ways: A well-developed
(patterned) surface has higher adsorption capacity,
whereas the presence of highly inclined regions (con-
straints) makes cell attachment more difficult. As follows
from the obtained data, initial biofilm formation on rough
surfaces occurs in two dimensions. If nutrient access is
limited by the configuration of surface constraints and/or
diffusion transport, bacteria can develop 3-D structures.
On the other hand, if the tested surface is plain and
smooth, bacteria always spread over it as a single-layer
(2-D) colony. Maturing of the biofilm and corresponding
total surface coverage lead to the development of three-
dimensional structures, which have been previously de-
scribed in the literature (4, 55, 56) and also observed in
our experiments.
10
10 100 1000 10000
100
1000
10000
100000
Time (sec)
No cells/mm
2
Figure 2. Effect of substratum type on the number of
Listeria monocytogenes which become attached. The substrata
are: ~aluminum; & polypropylene;
poly(ethyleneterephthalate);
B high-density polyethylene; ’ poly(vinyl chloride);
poly-
(tetrafluorethylene).
118 BIOFILM DEVELOPMENT ON PACKAGING MATERIALS
Kinetics of bacterial adhesion onto surface of selected
packaging materials is depicted in Figure 2. These results
support data in the literature that adhesion of Listeria
monocytogenes is material-dependent (34, 57).
FOODBORNE BIOFILMS AND ACTIVE (CONTROLLED-
RELEASE) PACKAGING MATERIALS
The food market has growing demand on fresh and mini-
mally processed foods. However, these foods are highly
perishable and more susceptible to microbial spoilage.
Thus, there is a strong need to develop new preservation
methods to achieve a required level of safety, quality, and
nutritional value of food during extended shelf-life period.
The use of active packaging (AP) materials is one of the
post-processing methods to preserve food products and
meet consumers’ expectations.
Antimicrobial packaging is designed to control micro-
bial growth in a food product. It consists of an antimicro-
bial agent (AMA) immobilized onto the internal surface of
a package or incorporated into packaging material (58). In
the latter case, AMA is released into a food product over
time. This permits us to extend shelf life of food products,
helping to reduce the amount of AMA in food formulation.
AP materials have many parameters that influence
their antimicrobial efficacy, which is thoroughly addressed
in a number of studies: polymer processing, polymer
morphology (59), polymer swelling (60), and AMA affinity
to the packaging material (61). In all these papers the food
product was considered as a homogeneous medium in full
contact with the packaging. However, surface morphology
of the foods is an important parameter that determines
mass transfer of an antimicrobial agent through the inter-
face between packaging and food.
Based on the nature of a food product and correspond-
ing morphology of a food surface, one can distinguish five
types of food-packaging contacts depicted in Figure 3.
If the surface of a food product is flat, direct contact
between the packaging and the food exists. This AP
system has maximum efficacy. An irregular food surface
will cause only partial contact between the packaging
material and the food product, developing noncontinuous
headspace. This influences AMA transport from the
packaging to the food. Depending on the dominating
physical state of a food product, the packaging surface
can be in contact with either solid or liquid food products,
sometimes both. These contacts can be direct or indirect if
headspace exists between the food surface and packaging
material (see Figure 4). Depending on the type of the food
product, the headspace can be liquid- or gas-filled.
Numerous parameters can influence the efficacy of AP,
including the packaging material properties, the antimi-
crobial transport, the bacterial population response and
the food matrix. Most of the published studies investi-
gated antimicrobial activity of the controlled release com-
pound by adding AMA directly to the foods or to the
packaging materials. However, these two methods have
significant disadvantages:
. When AMA is added directly to the food, obtained
data provide important information on the antimi-
crobial activity of AMA and its interaction with the
food matrix. But there is no time-dependent AMA
release; therefore these studies are insufficient for
the development of AP.
. On the other hand, if AMA is incorporated into the
packaging material, there is no control over the
antimicrobial release. The effects of packaging ma-
terial properties on the AMA release rate cannot be
distinguished from the effects of the AMA release
rate on the bacterial inhibition.
There is a need to understand bacterial response to the
AMA release without the influence of material-dependent
properties of AP. No standard method has been established
Packaging
Liquid
Liquid Gas
Packaging Packaging Packaging
Food
liquid
Food
solid
Food
solid
Food
solid
(a)(b)(c)(d)
Headspace
Figure 4. Dependence of a food/packa-
ging interface on a type of food product.
Packaging
Food
Food
Packaging
Food
Packaging
Food
Packaging
Food
Packaging
Headspace
(a)(b)(c)(d)(e)
Figure 3. Spatial organization of AP as a function of food surface morphology: direct contact (a), partial contact B1cm (b), B1mm (c),
B10 mm(d), and direct contact (e).
BIOFILM DEVELOPMENT ON PACKAGING MATERIALS 119
to investigate the effect of antimicrobial agent’s time-
dependent release on the bacterial response.
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BIOSENSOR TECHNOLOGY FOR FOOD
PACKAGING APPLICATIONS
PAUL TAKHISTOV
Department of Food Science,
Rutgers, the State University of
New Jersey New Brunswick,
New Jersy
This article provides an introduction to the emerging
technology of biosensors and its potential applications to
food packaging. This biosensor technology may used to
enhance the communication function of the intelligent
packaging system (see the Intelligent Packaging article)
to enhance the safety and quality of food products.
NEEDS OF FOOD QUALITY/SAFETY CONTROL
Food quality control is essential in the food industry;
nowadays, an efficient quality assurance is becoming
increasingly important. Consumers expect adequate qual-
ity of food product at a fair price, long shelf life, and high
product safety, whereas food inspectors require safe man-
ufacturing practices, adequate product labeling, and
compliance with the U.S. Food and Drug Administration
(FDA) regulations. Further more, food producers are
increasingly demanding the efficient control methods,
particularly through online or at-line quality sensors to
satisfy consumers’ and regulatory requirements and to
improve the feasibility of automated food processing and
quality of sorting. Also, food producers are demanding a
reduction in the production time (increase throughput)
and the final product cost.
Novel sensing technologies using biomaterials or nano-
materials can be used to detect quality and safety attri-
butes in packaged foods. These sensing technologies range
from rapid non destructive and noncontact to highly
specialized microsensing and nanobiosensing structures.
Micro- and nano-based sensors that use a variety of
transduction mechanisms to sense microbial and
BIOSENSOR TECHNOLOGY FOR FOOD PACKAGING APPLICATIONS 121
biochemical changes in food products are being explored.
Extensive development of biosensors for food safety and
quality control were stimulated by acquiring several new
food safety and key quality concepts during the last
decade, such as Hazard Analysis Critical Control Points
(see the HACCP article), Total Quality Management
(TQM), and ISO 9000 Certifications. The wave of terrorist
acts and foodborne disease outbreaks have raised the
importance of the food traceability and authentication
(1, 2). There are specific safety problems (pathogenic
micro-organisms, BSE, GMF, pollutants, etc.) that require
intensive control, data logging, and data treatments,
which can be controlled effectively only with the new
generations of biodetection systems (3). All these tasks
require rapid responce sensors for new integrated data
analysis systems and are an indispensable part of the
modern supply chain operation paradigm.
There are several possible sources of undesirable con-
taminations and/or changes in food products that can be
combined in five groups by their localization and occur-
rence. Three of them are food manufacturing-related as
follows: technology (processing and sequence of process
operations), industrial hygiene (food safety management
at the plant level and HACCP), and formulation (product
development, interactions of food additives/ingredients
with food matrix, and bioavailability). The sources of
food raw materials and their quality are the issue of
biosafety/biosecurity in the agricultural processing includ-
ing postharvesting technologies and logistics. The fifth
source of biohazards is the environment in the broadest
sense, which includes pollution, climate changes, and
anthropogenic environmental factors.
The major types of changes in foods are caused by the
sources of undesirable contaminants. They can be instru-
mentally controlled; hence, they represent the primary
targets for biosensors development and design. Indeed,
the great challenge is to develop the real-time and online
sensors and data systems suitable for surveying processes
and products, controlling automated processes and the
raw material stream, sensing the final products quality,
typing the product labels with nutritional and health
information, and much more. Today, the most important
quality parameters and concepts in food production con-
trol are as follows:
. Sensory: appearance, flavor, taste, texture, and
stability
. Nutritional: including health implications, such as
‘‘high in fiber,’’ ‘‘low cholesterol,’’ and ‘‘GMF free’’
. Composition and labeling: additives lists as well as
quality and ethical claims (e.g., ecological information)
. Pollutants record: environmental pollutants, veter-
inary drugs, agricultural chemicals, BSE-prions, and
mycotoxins
. Detection of foreign bodies: such as stones, glass or
metal fragments
. Microbial safety: in particular Listeria, Salmonella,
Campylobacter, Escherichia coli and Yersinia
. Shelf life: microbial, sensory, chemical, sterility test-
ing, and F
0
-values
. Production hygiene: cleaning and decontamination;
. HACCP: traceability and authentication
. Process parameters control: machine settings, tem-
perature, pressure, flow, aseptic conditions, and
many others
. Packaging: integrity, pinholes, gas permeability, and
migration control
BIOSENSORS: GENERAL FACTS
The potential susceptibility of the food supply chain to
natural or intentional contamination could result in com-
promised safety and quality of foods. Nano-bio sensors and
integrated microsystems could play a significant role of
detecting deteriorative changes in food packaging. In
modern food packaging development, appropriate sensing
technologies are required to detect substances in parts per
trillion for food safety, quality, and process control. The
development of new sensing devices may be achieved by
taking advantage of miniaturization of electronics and
nano-bio materials. These novel sensing systems can be
used to facilitate the online analysis of food stuffs.
Biosensor technology is a powerful alternative to con-
ventional analytical techniques, harnessing the specificity
and sensitivity of biological systems in small, low-cost
devices. Despite the promising biosensors developed in
research laboratories, there are not many reports of real
applications in packaging. A sensor is the device that can
detect a property or group of properties in a food product
and respond to it by a signal, often an electric signal. This
signal may provide direct information about the quality
factor(s) measured or may have known relation to the
quality factor. Usually, sensors are classified according to
their mode of use (see Figure 1).
Biosensors usually are small, analytical bioelectronic
devices that combine a transducer with a sensing biological
component (biologically active substance). The transducer,
Sensor Operation Mode
Off-line
1 hr -24 hr
At-line
< 30 min
On-line
< 5 min
Sensors are used,
in split-flow
measurements,
requiring reagent
additions,
sampling or
sufficient time for
the equilibration or
chemical reaction
to occur
Sensors/detectors
operate directly in
the process
stream, giving a
real-time signal
related to the
quality factor of
concern.
Sensors/assays
are used in the
laboratory with
extensive periods
of time required to
performing the
measurements
Figure 1. Sensor operation modes.
122 BIOSENSOR TECHNOLOGY FOR FOOD PACKAGING APPLICATIONS
which is in intimate contact with the biologically sensitive
material, can the measure weight, electrical charge, poten-
tial, current, temperature, or optical activity of the sub-
stance. The biologically active species include enzymes,
multienzyme systems, antibodies or antigens, receptors,
populations of bacterial or eukaryotic cells, or whole slices
of mammalian or plant tissue. Substances such as sugars,
amino acids, alcohols, lipids, nucleotides, and so on can
be specifically identified and their concentration measured
by these sensors. A schematic functional representation
of a biosensor and the detection principle is depicted in
Figure 2. The biosensor consists of a biological sensing
element integrated with a signal transducer; together, they
produce a reagent-free sensing system specific for the
target analyte. The biological component of a biosensor
used for the molecular detection is made of highly specia-
lized macromolecules or complex systems with the appro-
priate selectivity and sensitivity. Biosensors can be
classified according to the biocomponents used for the
detection.
The biodetection principle can be schematically de-
scribed as follows. A chemical, biological, or physical
sensor produces a signal (e.g., voltage, absorbance rate,
heat, or current) in response to a detectable event, such as
binding between two molecules. In case of a biological or
chemical sensor, this event typically involves a receptor
(e.g., macrocyclic ligand, enzyme, or antibody) binding to a
specific target molecule in a sample. Physical sensors, on
the contrary, measure the inherent physical parameters of
a sample, such as current or temperature, which can
change because of reactions occurring in it. In any case,
the signal is then transduced by passing it to a circuit
where it is digitized. The obtained digital information can
be stored in a memory, displayed on a monitor, or made
accessible via digital communications port.
Because it is essential that the sensor’s response be
detected, it is necessary that an appropriate transduction
mode for electrochemical signals, optical signals using
changes in the fluorescence or absorbance rate of a sample,
or plasmon resonance be available. With most sensors,
transduction is accomplished electrochemically or optically.
The transducer transforms the physicochemical varia-
tions occurring in the biosensing element as the result of a
positive detection event into an electric signal, which is
then amplified by an ad hoc designed electronic circuit and
used for the control of external devices. The transducers
can be electrochemical (amperometric, potentiometric,
and conductometric/impedimetric), optical, piezoelectric,
or calorimetric. Often, this classification is used to identify
the type of a biosensor (see Figure 3).
The bio-specific elements of the biosensor and transdu-
cer can be coupled together in one of the four possible ways
(4), that are schematically shown in Figure 4: membrane
entrapment, physical adsorption, matrix entrapment/por-
ous encapsulation, and covalent bonding.
In the membrane entrapment scheme, a semipermeable
membrane separates the analyte and the bio-element, and
the sensor is attached to the bioelement [collagen
Analyte
Bio-recognition
element
Transducer
Signal
processing
Data
analysis
Figure 2. Schematics of biodetection process.
Bio-recognition element
Principle of operation
Transducer
Biosensors
Optical Mechanical Electrochemical
Fluorescence
Surface plasmon
resonance
Adsorbance
Reflectance
Piezoelectric
Surface acoustic wave
Cantieliver resonance
frequency
Amperometric
Potentiometric
Impedimetric
Molecular Cell-based Tissue-based
Figure 3. Biosensors classification.
BIOSENSOR TECHNOLOGY FOR FOOD PACKAGING APPLICATIONS 123
membranes, synthetic pre-activated membranes (5), and
cellulose-acetate membranes]. The physical adsorption
scheme depends on a combination of van der Waals forces,
hydrophobic forces, hydrogen bonds, and ionic forces to
attach the biomaterial to the sensor surface (6). The porous
entrapment scheme is based on forming a porous encapsu-
lation matrix around the biological material that helps in
binding it to the sensor [nylon net (7), carbon paste (8), or
graphite composites (9)]. In the case of covalent bonding,
the sensor surface is treated as a reactive group to which
the biological material can bind (10). One of the bioselective
elements most frequently used in biosensors is an enzyme.
These large protein molecules act as catalysts in chemical
reactions but remain themselves unchanged at the end of
reaction.
Mechanical (Resonant) Biosensors. In this type of bio-
sensors, an acoustic wave transducer is coupled with an
antibody (biosensitive element). When the analyte mole-
cules (antigens) attach to the membrane (cantilever), the
membrane mass changes, resulting in a subsequent
change in the resonant frequency of the transducer (11).
This frequency change is detected and measured (4).
Sensors Based on Electromagnetic Waves. Electromag-
netic sensors may be classified by the wavelength of
the electromagnetic waves they use: visible (VIS)
(400–700 nm), ultraviolet (10–400 nm), infrared [700–
30,000 nm: nearinfrared (NIR (12), FTIR (13), MRI (14)]
waves, microwaves (15) (1–10 cm), radiofrequency (16)
(1–10 m), and X-rays (17) (100 pm
1
nm). Each sensor
class may be subdivided even more according to the
molecular information that can be obtained through the
interaction. For instance, infrared sensors may be sub-
divided into near-infrared (700–2500 nm), mid-infrared
(2500–30,000 nm), far-infrared (up to 1,000,000 nm), and
thermography (1–15 mm) sensors, which all extract differ-
ent information from the molecules (sample) interacting
with the waves. We may also classify these sensors
according to their precise type of interaction: absorbance,
transmittance, or reflectance of light.
Sensors based on interactions with electromagnetic
radiation waves have been on the market for many years,
in particular for laboratory purposes. Online examples of
such sensors are also numerous: X-rays used for foreign
body detection (18), visible light sensors for color recogni-
tion or machine vision inspections (19), near-infrared
sensors for quality inspection and temperature measure-
ments (20), or microwave sensors for the detection of
water content (21).
Optical Detection Biosensors. Strictly speaking, optical
biosensors belong to the larger class of electromagnetic
detectors, but because of their importance and broad use,
they are usually considered as a separate group of bio-
sensitive devices. The output signal measured in this type
of biosensors is a light signal (22). These biosensors can be
made based on optical diffraction or electrochemilumines-
cence (23).
Surface Plasmon Resonance (SPR). SPR is another opti-
cal phenomenon used in new sensors, often in those that
involve antibodies or enzymes. The optical range used is
most often in the visible part of the spectrum, but it may
also be in the NIR range. Traditionally, SPR devices detect
minute changes in the refractive index of the sensing
surface and its immediate vicinity. They may detect these
changes by a diffraction grating, with a prism on a glass
slide, or through an optical waveguide carrying a thin
metal layer (gold). The metal layer carries a sensitizing
layer (e.g., immobilized antibodies or other molecules
binding the analyte specifically; this layer is in contact
with the sample). Inside the device, a collective excitement
of electrons in the metal film occurs and leads at a specific
wavelength to a total absorption of light at a particular
angle of incidence. This angle depends on the refractive
indices on either side of the metal film. Specific molecules
binding to the sensitizing layer change the refractive
index, which changes an angle of total absorption; this
angle is measured and correlated to the concentration of
the analyte.
The SPR detection technique has been used by Hell-
naes (24) for online and at-line detection of veterinary
drug residues (hormones and antibiotics) in dairies and
slaughterhouses. Clenbuterol and ethinyl-estradiol in bo-
vine urine, sulfamethazine (SMT), and sulfadiazine (SDZ)
in porcine bile, as well as SMT, SDZ, and enrofloxacin in
milk have been successfully detected by the technique.
The developed biosensor operates in real time and can
simultaneously detect up to 8 different veterinary drugs
with a throughput of up to 600 samples per day. The
project participants have established a new company to
produce and develop the sensor systems, and several new
and elegant designs of SPR sensors are now under study.
The SPR sensor principle has also been used by Patel (25).
The sensor developed as a result of this research has been
applied to the quantification of mycotoxins, Listeria, and
SENSOR
SENSOR
SENSOR
SENSOR
Covalent bonding
Matrix entrapment
Physical adsorption
Membrane entrapment
Figure 4. Coupling of biorecognition element (8) with the trans-
ducer on a sensor substrate.
124 BIOSENSOR TECHNOLOGY FOR FOOD PACKAGING APPLICATIONS
markers for growth hormones [recombinant bovine soma-
totrophin (rBST)].
Most current research is focused on the NIR/VIS
sensors, SPR sensors and nuclear magnetic resonance
(NMR) sensors (pulsed and low resolution); some work
has also been done on fluorescence sensors, MIR and
Raman sensors, Fourier Transform NIR sensors, thermo-
graphy-based sensors, and sensors that combine two or
more sensor principles.
Electrochemical Biosensors. Electrochemical biosensors
are mainly used for the detection of hybridized DNA,
DNA-binding drugs, glucose concentration, and so on.
The underlying principle of these biosensors is that
many chemical reactions produce or consume ions or
electrons that in turn cause some changes in the electrical
properties of the solution; these changes can be sensed
out and measured (26). The electrochemical biosensor can
be classified based on the measured electrical parameter
as conductimetric, amperometric, or potentiometric (27).
Impedimetric/Conductometric Biosensors. Many biologi-
cal processes involve changes in the concentrations of
ionic species. Such changes can be used by biosensors,
which detect changes in electrical conductivity. The mea-
sured parameter is the electrical conductance/resistance
of the solution. When electrochemical reactions produce
ions or electrons, the overall conductivity/resistivity of
the solution changes (28). This change is measured and
calibrated to a proper scale. Conductance measurements
have relatively low sensitivity. The electric field is gener-
ated using sinusoidal voltage, which helps in minimizing
undesirable effects such as Faradaic process, double-layer
charging, and concentration polarization (29).
Impedimetric biosensors use changes in the electrical
conductivity in the frequency domain (impedance) of a
biological system for sensing and detection (6, 30, 31).
Impedance spectroscopy provides a powerful tool for
investigating a variety of bioelectric processes for both
electrical and nonelectrical applications. In impedance
spectroscopy, the current flowing through a sample cell
that contains a nano-scale patterned bio-interface and the
voltage across this cell are measured as a function of
frequency (32–34). The design of impedimetric sensors is
similar to conductivity-based sensors (26, 35–39). Enzyme/
antibody immobilization on electrode surface makes these
sensors highly selective and sensitive (40).
Amperometric Biosensors. This highly sensitive biosen-
sor can detect electro-active species present in biological
test samples. Enzyme-catalyzed redox reactions can form
the basis of a major class of biosensors if the flux of redox
electrons can be determined (41). Normally, a constant
voltage is applied between two electrodes, and the current,
which is caused by the electrode reaction, is determined.
The first and simplest biosensor was based on this prin-
ciple. It was for the determination of glucose and made use
of the Clark oxygen electrode. In the case of amperometric
biosensors, the measured parameter is an electric current.
Some of the most recent applications of amperometric
biosensors include glucose sensor for meat freshness
(43); glucose sensor for use in fermentation systems (43);
rapid cell number monitor (44); monitor for herbicides in
surface waters (45, 46); amperometric enzyme-linked im-
munosorbent assay (ELISA) method based on the self
enzyme amplification system (36); amperometric and no-
vel fluorescent DNA probes (47).
Potentiometric Biosensors. In this type of sensors, the
measured parameter is the oxidation/reduction potential
of an electrochemical reaction (48). The simplest potentio-
metric technique is based on the concentration depen-
dence of the potential, E, at reversible redox electrodes
according to the Nernst equation (29) E ¼ E
0
þ
RT
nF
ln a
s
;
where E
o
is the standard redox potential, R is the gas
constant, T is the absolute temperature, F is the Faraday
constant, n is the number of exchanged electrons of the
substance S, and a
s
is the activity of the substance S.
Changes in ionic concentrations are easily determined by
use of ion-selective electrodes (48). This forms the basis of
potentiometric biosensors (30). Many biocatalyzed reac-
tions involve charged species each of which will absorb or
release hydrogen ions according to their pK
a
and the pH of
the environment (49). This allows a relatively simple
electronic transudction using the most common ion-selec-
tive electrode, which is the pH electrode (50).
Field Effect Transistors (FETs) and Ion-Selective Field Effect
Transistors (ISFETs). Potentiometric biosensors can be min-
iaturized by the use of FET. ISFETs are low-cost devices
that are in mass production (51). A recent development
from ion-selective electrodes is the production of ISFETs
and their biosensor use as enzyme-linked field effect
transistors (ENFETs). Enzyme membranes are coated
on, the ion-selective gates of these electronic devices, the
biosensor responding to the electrical potential change via
the current output. Thus, these are potentiometric de-
vices, although they directly produce changes in the
electric current. Reference shows a diagrammatic cross-
section through an npn hydrogen ion responsive ISFET
with a biocatalytic membrane. The buildup of positive
charge on this surface (the gate) repels the positive holes
in the p-type silicon causing a depletion layer and allowing
the current to flow. In Abdelmalek et al. (52), Langmuir-
Blodgett films that contain butyrylcholinestrase (BuChE)
are fabricated to realize an ISFET for the detection of
organophosphorus pesticides in water.
Cell-Based Biosensors. Cell-based biosensors have been
implemented using micro-organisms, particularly for en-
vironmental monitoring of pollutants (53). Biosensors that
incorporate mammalian cells have a distinct advantage of
responding in a manner that can offer insight into the
physiological effect of an analyte (54, 55). Several ap-
proaches for the transduction of cellular signals (56) are
described in the literature: measures of cell metabolism,
impedance (57), intracellular potentials, and extracellular
potentials (58). Among these approaches, networks of
excitable cells cultured on microelectrode arrays (53, 54,
59–61) are uniquely poised to provide rapid, functional
classification of an analyte and ultimately constitute a
potentially effective cell-based biosensor technology.
BIOSENSOR TECHNOLOGY FOR FOOD PACKAGING APPLICATIONS 125
Keese and Giaever (62) have designed a biosensor that can
be used to monitor cell morphology in tissue culture
environment. The sensing principle used is known as
electric cell-substrate impedance sensing (ECIS). In this
process, a small gold electrode is immersed into the tissue
culture medium. After cells attach and spread over the
electrodes, the electric impedance measured across the
electrode chamber changes. These changes in impedance
can be used for understanding cell behavior in the culture
medium. The attachment and spreading of the cells are
important factors for successful use of this biosensor.
Unfortunately, some types of cells, e.g., cancerous cells,
can grow and reproduce freely in a medium without being
attached to any substrate/surface, that makes them im-
possible to detect with these sensors.
Cornell et al. (63) proposed biosensor mimics biological
sensory functions and can be used with most types of
receptor, including antibodies and nucleotides. The tech-
nique is flexible and even in its simplest form it is
sensitive to pico-molar concentrations of proteins.
Lab-on-a-Chip Systems and DNA Detection Devices. Sig-
nificant advances have been made in the development of
micro-scale technologies for biomedical and drug discov-
ery applications. The first generation of microfluidics-
based analytical devices [Lab-on-a-Chip (64)] have been
designed and are already functional. Microfluidic devices
offer unique advantages in sample handling (65–67),
reagent mixing (68–70), separation (71–73), and detection
(74). These devices include, but are not limited to are
devices for cell sampling (75), cell trapping and cell sorting
devices (59, 76–79), flow cytometers (67, 80, 81), devices
for cell treatment: cell lysis, poration/gene transfection,
and cell fusion devices (82).
Biosensors used for DNA detection are used to identify
small concentrations of DNA (of micro-organisms such as
viruses or bacteria) in a large sample. The detection relies
on comparing sample DNA with a DNA of known micro-
organism (probe DNA) (83). Because the sample solution
may contain only a small number of micro-organism
molecules, multiple copies of the sample DNA need to be
created for proper analysis (84). This is achieved with an
aid of the polymerase chain reaction (PCR). PCR starts by
splitting the sample’s double-helix DNA into two parts by
heating it. If the reagents contain proper growth enzymes,
then each of these strands would grow the complementary
missing part and form the double-helix structure again.
This happens after the temperature is lowered. Thus, in
one heating/cooling cycle, the amount of sample DNA is
doubled (85). In general, PCR is power consuming, so
previously it was not possible to fabricate portable biode-
tectors that can perform PCR. But, using newly developed
MEMS devices, such biodectors (also known as lab-on-a-
chip systems) have been created. In these MEMS-based
devices the amount of reagent used is scaled down (86).
DNA-Based Sensors/Assays. The general principle of
DNA probe assay is similar to the immunoassay described
in. Indeed, even the applications of DNA probes and
monoclonal antibody immunoassay frequently overlap,
thus establishing a ‘‘competition’’ between the two possi-
ble approaches.
One of the most important applications for DNA probes
is the testing for virus infections (87). For probes of
infectious disease, it is assumed that all strains can still
contain a common DNA sequence region and thus be
identified by a single probe. Recognized by the cell as a
foreign body, viruses will induce an antigenic reaction
causing antibody generation so they can also be detected
in an immunoassay (88).
Another type of biosensor developed by the Naval
Research Laboratory (85) uses magnetic field instead of
optics or fluorescence. This sensor equipped with magnetic
sensors and microbeads (89) can detect the presence and
concentration of bioagents. The magnetic sensor (group of
sensors) is coated with single-stranded DNA probes spe-
cific for a given bioagent or sample DNA. Once a single
strand of DNA probe and a single strand of sample DNA
find each other, they form a double-stranded (double-
helix) structure, which in turn binds a single magnetic
microbead. When a magnetic bead is present on a sensor
surface, its resistance decreases, which can be detected
and measured.
BIOSENSORS AND FOOD PACKAGING
Integrated Sensor-Packaging Systems
The basic function of sensors-packaging system is to
monitor the package environment, product state/status,
and perform data exchange with the external databases,
providing information for decision making. Intelligent
packaging is the integrated system of a complex structure
with discrete/distributed sensor and data carrier ele-
ments. Technical realization of such a packaging system
includes integration of sensitive elements into the packa-
ging materials and/or labels, and integration of sensors’
data into the intelligent packaging information flow, i.e.,
data layer. Such integration allows to perform online and
‘‘on-shelf’’ control of the internal and/or external package
environments. Biosensors can be created by chemical
modification and sensibilizing of packaging labels, embed-
ding microdevices (‘‘smart chips’’), or depositing micro-
and nano-sensitive elements into the label or the package
itself. In the future, this system can be used to trigger the
controllable release of bioactive components and targeted
delivery of protective agents into the food product. One of
the most important elements of the Internet Protocol (IP)
concept is integration of sensors and detectors into the
package itself. Examples of monitoring activities include
visual observations/control and measurements of tem-
perature, time, pH, and moisture levels. In the future,
this system can be used to trigger the controllable release
of bioactive components and the targeted delivery of
protective
agents into the
food product.
Based on the type of the sensor and their location, it is
possible to recognize three major types of sensor-packa-
ging systems (see Figure 5):
. Off-package — remote sensing devices without re-
quirements of direct contact with the package
126 BIOSENSOR TECHNOLOGY FOR FOOD PACKAGING APPLICATIONS
materials of packaging content, i.e., various spectro-
scopic devices (Raman, IR, NIR, and radiofrequensy).
. On-package — truly integrated devises that located
on the packaging itself. RFID devices can be related
to this category. Sensing packaging materials, which
are sensors with distributed parameters, also belong
to this type of sensors.
. In-package — sensing device is placed inside of the
package. In some cases sensor can be part of the
product formulation.
Integrated Sensor-Packaging Systems May Enhance Food
Safety
Existing food distribution network includes producers,
logistic operators, and processors. Timely detection of
unsafe foods entering this network is the main issue
that food safety system should address. The HACCP
system, which has become the industrial standard for
food safety management is designed to identify health
hazards and to establish strategies and procedures to
prevent, eliminate, or reduce their occurrence. Each stage
of the food products flow is individually controlled, but
there is no integrated food safety system that combines
material and informational flows into one continuous food
safety management process. There is a need to enhance
the existing HACCP procedures by integration of material
and information flows and to ensure early detection of
deliberate food contamination at any point along the
production pathway. In general the intelligent packaging
framework is perfectly adapted to be used in the HACCP
workflow and uses the principle of information system
cyclic interactions with the environment (Figure 6).
The intelligent packaging framework enhances the
existing HACCP system by incorporating its virtual data
layer into the food safety management structure. Intelli-
gent packaging can be integrated into the HACCP system
as a data carrier, which delivers proper information about
product history, establishes critical limits, and acts as a
self-reporting data tag that creates natural link with an
external knowledge base. Hence, the Critical Control
Points that implement elements of an intelligent packa-
ging will receive new functionality. Combining data of
product properties and proper storage regimes with the
FOOD PRODUCT
PACKAGING
off-package
sensor
on-package
sensor
oi-package
sensor
Discrete
Distributed
Discrete Distributed
Figure 5. Sensor-packaging systems.
Material Flow
Information Flow
Advanced logistic
Management System
Packaging Flow
sensors
Figure 6. An integration of sensors into infor-
mation/material flow in distribution network.
BIOSENSOR TECHNOLOGY FOR FOOD PACKAGING APPLICATIONS 127